1. Developmental Biology

A zebrafish embryo screen utilizing gastrulation identifies the HTR2C inhibitor pizotifen as a suppressor of EMT-mediated metastasis

  1. Joji Nakayama  Is a corresponding author
  2. Lora Tan
  3. Yan Li
  4. Boon Cher Goh
  5. Shu Wang
  6. Hideki Makinoshima
  7. Zhiyuan Gong  Is a corresponding author
  1. Department of Biological Science, National University of Singapore, Singapore
  2. Cancer Science Institute of Singapore, National University of Singapore, Singapore
  3. Tsuruoka Metabolomics Laboratory, National Cancer Center, Japan
  4. Shonai Regional Industry Promotion Center, Japan
  5. Institute of Bioengineering and Nanotechnology, Singapore
  6. Division of Translational Research, Exploratory Oncology Research and Clinical Trial Center, National Cancer Center, Japan
https://doi.org/10.7554/eLife.70151
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Cite this article
  1. Joji Nakayama
  2. Lora Tan
  3. Yan Li
  4. Boon Cher Goh
  5. Shu Wang
  6. Hideki Makinoshima
  7. Zhiyuan Gong
(2021)
A zebrafish embryo screen utilizing gastrulation identifies the HTR2C inhibitor pizotifen as a suppressor of EMT-mediated metastasis
eLife 10:e70151.
https://doi.org/10.7554/eLife.70151
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5 figures, 8 tables and 5 additional files

Figures

Figure 1 with 2 supplements
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A chemical screen for identification of epiboly-interrupting drugs.

(A) Cumulative results of the chemical screen in which each drug was used at either 10 µM (left) or 50 µM (right) concentrations. 1280 FDA, EMA, or other agencies-approved drugs were subjected to this screening. Positive ‘hit’ drugs were those that interrupted epiboly progression. (B) Representative samples of the embryos that were treated with indicated drugs.

Figure 1—figure supplement 1
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Gene expression profiles obtained from zebrafish embryos at either 50%-epiboly (top left), shield (top right), or 75%-epiboly stage (bottom left) were analyzed based on the hallmark gene sets derived from the Molecular Signatures Database (MSigDB) (Liberzon et al., 2015).

The zebrafish transcriptomic data was sourced from White et al., 2017 eLife (Subramanian et al., 2005). Gene sets that were significantly enriched (FDR < 0.25) were presented with the normalized enrichment score (NES) and nominal p value. Source data files for analysis of either gene expression and enriched pathways are uploaded as gene set enrichment analysis (GSEA) Source data 1 and 2, respectively.

Figure 1—figure supplement 2
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Epiboly could serve as a marker for this screening.

(A) Western blot analysis of protein arginine methyltransferase 1 (PRMT1) (upper left) and cytochrome P450 family 11 (CYP11A1) (middle left) protein levels in non-metastatic human cancer cell line (MCF7) and highly metastatic human cancer cell lines (MDA-MB-231, MDA-MB-435, MIA-PaCa2, PC9, HCCLM3, PC3, and SW620); β-actin loading control is shown (bottom left). Preliminary experiments confirmed that epiboly could serve as a marker for this screening assay. Quantification analyses of western blotting bands. The analyses were performed by ImageJ. Signal strength of bands of PRMT1 (Top right) and CYP11A1 (bottom right) was normalized by that of β-actin. (B) Knockdown of PRMT1 or CYP11A1 in MDA-MB-231 cells. MBA-MB-231 cells were transfected with a control short hairpin RNA (shRNA) targeting LacZ, and one of four independent shRNAs targeting PRMT1 (clones #1 to #4), or one of two independent shRNAs targeting CYP11A1 (clones #1 to #4). Reduced PRMT1 and CYP11A1 expression, determined by western blot, in sub-cell lines of MDA-MB-231 cells expressing PRMT1 shRNA (clones #3 and #4) or CYP11A1 (clones #2 and #4), compared with controls (parental cell line MDA-MB-231 and control shRNA cells); β-actin levels shown as a loading control. Quantification analyses of western blotting bands. The analyses were performed by ImageJ. Signal strength of bands of PRMT1 (top right) and CYP11A1 (bottom right) was normalized by that of β-actin. (C) Effect of shRNAs targeting either PRMT1 or CYP11A1 on cell motility and invasion of MBA-MB-231 cells. Parental MDA-MB-231 cells and four sub-cell lines of MDA-MB-231 cells that were transfected with either shRNA targeting either LacZ, two independent shRNAs targeting PRMT1 (clones #3 and #4) or two independent shRNAs targeting CYP11A1 (clones #2 and #4) were subjected to Boyden chamber assays. (D) Zebrafish embryos treated with either vehicle (DMSO), 10 μM niclosamide, or 50 µM vinpocetine. Approximately 20 embryos were treated with either DMSO as a vehicle control, niclosamide, or vinpocetine. The treatment was started at 4 hours post fertilization (hpf) when all of embryos reached sphere stage and ended at 9 hpf when control embryos reached 80–90% epiboly stage. Each experiment was performed at least twice. Statistical analysis was determined by Student’s t test.

Figure 2 with 2 supplements
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Pizotifen, one of epiboly-interrupting drugs, suppressed metastatic dissemination of human cancer cells lines in vivo and vitro.

(A) Effect of the epiboly-interrupting drugs on cell motility and invasion of MBA-MB-231 cells. MBA-MB-231 cells were treated with vehicle or each of the epiboly-interrupting drugs and then subjected to Boyden chamber assays. Fetal bovine serum (1% v/v) was used as the chemoattractant in both assays. Each experiment was performed at least twice. (B) Western blot analysis of HTR2C levels (top) in a non-metastatic human cancer cell line, MCF7 (breast) and highly metastatic human cancer cell lines, MDA-MB-231 (breast), MDA-MB-435 (melanoma), PC9 (lung), MIA-PaCa2 (pancreas), PC3 (prostate), and SW620 (colon); GAPDH loading control is shown (bottom). (C) Effect of pizotifen on cell motility and invasion of MBA-MB-231, MDA-MB-435, and PC9 cells. Either vehicle or pizotifen treated the cells were subjected to Boyden chamber assays. Fetal bovine serum (1% v/v) was used as the chemoattractant in both assays. Each experiment was performed at least twice. (D) and (E) Representative images of dissemination of 231R, shLacZ 231R or shHTR2C 231R cells in zebrafish xenotransplantation model. The fish larvae that were inoculated with 231R cells were treated with either vehicle (top left) or the drug (lower left) (D). The fish larvae that were inoculated with either shLacZ 231R or shHTR2C 231R cells (lower left) (E). White arrows head indicate disseminated 231R cells. The images were shown in 4× magnification. Scale bar, 100 µm. The mean frequencies of the fish showing head, trunk, or end-tail dissemination were counted (graph on right). Each value is indicated as the mean ± SEM of two independent experiments. Statistical analysis was determined by Student’s t test.

Figure 2—figure supplement 1
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Blocking Dopamine receptor D2 with S(-) Eticlopride hydrochloride suppressed cell motility and invasion of highly metastatic human cancer cells in a dose-dependent manner.

(A) Quantification analyses of western blotting bands in Figure 2B. The analyses were performed by ImageJ. Signal strength of bands of HTR2C (left) and DRD2 (right) was normalized by that of GAPDH. (B) Western blot analysis of DRD2 levels in non-metastatic human cancer cell line, MCF7 (breast) and highly metastatic human cancer cell lines, MDA-MB-231 (breast), MDA-MB-435 (melanoma), MIA-PaCa2 (pancreas), PC3 (prostate), and SW620 (colon); GAPDH loading control is shown (bottom). GAPDH control was obtained in the same experiment from Figure 2B. (C) Effect of S(-)eticlopride hydrochloride on cell motility and invasion of MBA-MB-231, MDA-MB-435, and PC9 cells. Either vehicle- or pizotifen-treated cells were subjected to Boyden chamber assays. Fetal bovine serum (1% v/v) was used as the chemoattractant in both assays. Each experiment was performed at least twice. Statistical analysis was determined by Student’s t test.

Figure 2—figure supplement 2
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Pizotifen suppressed metastatic dissemination of MDA-MB-231 and MIA-PaCa2 cells in a zebrafish xenotransplantation model.

(A) Representative images of dissemination of 231R cells in zebrafish xenotransplantation model. The fish larvae that were inoculated with 231R cells were treated with either vehicle (top left, bottom left) or pizotifen (top right, bottom right). (B) Representative images of dissemination of MIA-PaCa2 cells in zebrafish xenotransplantation model. The fish were inoculated with MIA-PaCa2 cells, and treated with either vehicle (top left) or drug (lower left). White arrow heads indicate disseminated MIA-PaCa2 cells. The images were shown in 4× magnification. Scale bar, 100 μm. The mean frequencies of the fish showing head, trunk, or end-tail dissemination were tabulated (right). Each value is indicated as the mean ± SEM of two independent experiments. Statistical analysis was determined by Student’s t test.

Figure 3 with 1 supplement
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Pizotifen suppressed metastatic progression in a mouse model of metastasis.

(A) Mean volumes (n = 10 per group) of 4T1 primary tumors formed in the mammary fat pad of either vehicle- or pizotifen-treated mice at day 10 post injection. (B) Ki67 expression level in 4T1 primary tumors formed in the mammary fat pad of either vehicle- or pizotifen-treated mice at day 10 post injection. The mean expression levels of Ki67 (n = 10 mice per group) were determined and were calculated as the mean ration of Ki67-positive cells to 4’,6-diamidino-2-phenylindole (DAPI) area. (C) Representative images of primary tumors on day 10 post injection (top panels) and metastatic burden on day 70 post injection (bottom panels) taken using an IVIS Imaging System. (D) Representative images of the lungs from either vehicle- (top) or pizotifen-treated mice (bottom) at 70 days post tumor inoculation. Number of metastatic nodules in the lung of either vehicle- or pizotifen-treated mice (right). (E) Representative hematoxylin and eosin (H&E) staining of the lung (top) and liver (bottom) from either vehicle- or pizotifen-treated mice. Black arrow heads indicate metastatic 4T1 cells. (F) The mean number of metastatic lesions in step sections of the lungs from the mice that were inoculated with 4T1-12B cells expressing short hairpin RNA (shRNA) targeting for either LacZ or HTR2C. (G) Representative H&E staining of the lung and liver from the mice that were inoculated with 4T1-12B cells expressing shRNA targeting for either LacZ or HTR2C. Black arrow heads indicate metastatic 4T1 cells. Each value is indicated as the mean ± SEM. Statistical analysis was determined by Student’s t test.

Figure 3—figure supplement 1
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Cleaved caspase 3 expression level in 4T1 primary tumors formed in the mammary fat pad of either vehicle- or pizotifen-treated mice at day 10 post injection.
Figure 4 with 1 supplement
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HTR2C induced epithelial-to-mesenchymal transition (EMT)-mediated metastatic dissemination of human cancer cells.

(A) The morphologies of the MCF7 and HaCaT cells expressing either the control vector or HTR2C were revealed by phase contrast microscopy. (B) Immunofluorescence staining of E-cadherin, EpCAM, vimentin, and N-cadherin expressions in the MCF7 cells from A. (C) Expression of E-cadherin, EpCAM, vimentin, N-cadherin, and HTR2C was examined by western blotting in the MCF7 and HaCaT cells; GAPDH loading control is shown (bottom). (D) Effect of HTR2C on cell motility and invasion of MCF7 cells. MCF7 cells were subjected to Boyden chamber assays. Fetal bovine serum (1% v/v) was used as the chemoattractant in both assays. Each experiment was performed at least twice. (E) Representative images of dissemination patterns of MCF7 cells expressing either the control vector (top left) or HTR2C (lower left) in a zebrafish xenotransplantation model. White arrow heads indicate disseminated MCF7 cells. The mean frequencies of the fish showing head, trunk, or end-tail dissemination tabulated (right). Each value is indicated as the mean ± SEM of two independent experiments. Statistical analysis was determined by Student’s t test.

Figure 4—figure supplement 1
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HTR2C promoted EMT-mediated metastatic dissemination of poorly metastatic human cancer cells in a zebrafish xenotransplantation model.

(A) Quantification analyses of western blotting bands in Figure 4C. The analyses were performed by ImageJ. Signal strength of bands of E-cadherin, EpCAM, N-cadherin, vimentin, ZEB1, and HTR2C was normalized by that of GAPDH. (B) Representative images of dissemination patterns of MCF7 cells expressing either the control vector (top left, middle left, bottom left) or HTR2C (top right, middle right, bottom right) in a zebrafish xenotransplantation model.

Figure 5 with 5 supplements
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Pizotifen restored mesenchymal-like traits of MDA-MB-231 cells into epithelial traits through blocking nuclear accumulation of β-catenin.

(A) Immunofluorescence (IF) staining of E-cadherin in either vehicle- or pizotifen-treated MDA-MB-231 cells. (B) Surface expression of E-cadherin in either vehicle (black)- or pizotifen (red)-treated MDA-MB-231 cells by FACS analysis. Non-stained controls are shown in gray. (C) Protein expressions levels of E-cadherin, ZEB1, and β-catenin in the cytoplasm and nucleus of 4T1 primary tumors from either vehicle- or pizotifen-treated mice are shown; Luciferase, histone H3, and β-tubulin are used as loading control for whole cell, nuclear, or cytoplasmic lysate, respectively. (D) Protein expression levels of epithelial and mesenchymal markers and ZEB1 in either vehicle- or pizotifen-treated MDA-MB-231 cells or E-cadherin-positive (E-cad+) cells in pizotifen-treated MDA-MB-231 cells are shown. (E) IF staining of β-catenin in the MCF7 cells expressing either vector control (top left, bottom left) or HTR2C (top right, bottom right). (F) Expressions of β-catenin in the cytoplasm and nucleus of MCF7 cells. (G) IF staining of β-catenin in either vehicle (top left, bottom left) or pizotifen-treated MDA-MB-231 cells (top right, bottom right). (H) Expressions of β-catenin in the cytoplasm and nucleus of MDA-MB-231 cells and the E-cad+ cells.

Figure 5—figure supplement 1
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Quantification analyses of western blotting bands in Figure 5C.

The analyses were performed by ImageJ. Signal strength of bands of E-cadherin, EpCAM, p-GSK-3β, GSK-3β were normalized by that of luciferase. Signal strength of bands of nuclear and cytoplasmic β-catenin was normalized by that of histone H3 and β-tubulin, respectively.

Figure 5—figure supplement 2
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Quantification analyses of western blotting bands in Figure 5D.

The analyses were performed by ImageJ. Signal strength of bands of E-cadherin, EpCAM, KRT18, KRT19, MMP1, MMP3, vimentin, and S100A4 was normalized by that of GAPDH.

Figure 5—figure supplement 3
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Quantification analyses of western blotting bands in Figure 5F.

The analyses were performed by ImageJ. Signal strength of bands of nuclear and cytoplasmic β-catenin was normalized by that of histone H3 and β-tubulin, respectively. Signal strength of bands of p-GSK-3β and GSK-3β was normalized by that of GAPDH.

Figure 5—figure supplement 4
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Expression of Snail and Twist1 was examined by western blotting in the MCF7 cells (left); GAPDH loading control is shown (bottom).

Protein expression levels of Twist1 in either vehicle- or pizotifen-treated MDA-MB-231 cells are shown (middle): GAPDH loading control is shown (bottom). Protein expression levels of Snail and Twist1 of 4T1 primary tumors from either vehicle- or pizotifen-treated mice are shown (right); luciferase is used as loading control for whole cell. Luciferase control was obtained in the same experiment from Figure 5C.

Figure 5—figure supplement 5
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Quantification analyses of western blotting bands in Figure 5H.

The analyses were performed by ImageJ. Signal strength of bands of nuclear and cytoplasmic β-catenin was normalized by that of histone H3 and β-tubulin, respectively. Signal strength of bands of p-GSK-3β and GSK-3β was normalized by that of GAPDH.

Tables

Table 1
A list of the genes that are involved between gastrulation and metastasis progression.

A list of the 50 genes that play essential role in governing both metastasis and gastrulation progression. The gastrulation defects in Xenopus or zebrafish that are induced by knockdown of each of these genes were indicated. The molecular mechanism in metastasis that is inhibited by knockdown of each of the same genes was indicated.

GenesGastrulation defectsRefEffects in metastasisRef
BMPConvergence and extensionKondo, 2007EMTKatsuno et al., 2008
WNTConvergence and extensionTada and Smith, 2000Migration and invasionVincan and Barker, 2008
FGFConvergence and extensionYang et al., 2002InvisionNomura et al., 2008
EGFEpibolySong et al., 2013MigrationLu et al., 2001
PDGFConvergence and extensionDamm and Winklbauer, 2011EMTJechlinger et al., 2006
CXCL12Migration of endodermal cellsMizoguchi et al., 2008Migration and invasionShen et al., 2013
CXCR4Migration of endodermal cellsMizoguchi et al., 2008Migration and invasionShen et al., 2013
PIK3CAConvergence and extensionMontero et al., 2003Migration and invasionWander et al., 2013
YESEpibolyTsai et al., 2005MigrationBarraclough et al., 2007
FYNEpibolySharma et al., 2005Migration and invasionYadav and Denning, 2011
MAPK1EpibolyKrens et al., 2008MigrationRadtke et al., 2013
SHP2Convergence and extensionJopling et al., 2007MigrationWang et al., 2005
SNAI1Convergence and extensionIp and Gridley, 2002EMTBatlle et al., 2000
SNAI2Mesoderm and neural crest formationShi et al., 2011EMTMedici et al., 2008
TWIST1Mesoderm formationCastanon and Baylies, 2002EMTYang et al., 2004
TBXTConvergence and extensionTada and Smith, 2000EMTFernando et al., 2010
ZEB1EpibolyVannier et al., 2013EMTSpaderna et al., 2008
GSCMesodermal patterningSander et al., 2007EMTHartwell et al., 2006
FOXC2Unclear, defects in gastrulationWilm et al., 2004EMTMani et al., 2007
STAT3Convergence and extensionMiyagi et al., 2004MigrationAbdulghani et al., 2008
POU5F1EpibolyLachnit et al., 2008EMTDai et al., 2013
EZH2Unclear, defects in gastrulationO’Carroll et al., 2001InvasionRen et al., 2012
EHMT2Defects in neurogenesisLin et al., 2005Migration and invasionChen et al., 2010
BMI1Defects in skeleton formationvan der Lugt et al., 1994EMTGuo et al., 2011
RHOAConvergence and extensionZhu et al., 2006Migration and invasionYoshioka et al., 1999
CDC42Convergence and extensionChoi and Han, 2002Migration and invasionReymond et al., 2012
RAC1Convergence and extensionHabas et al., 2003Migration and invasionVega and Ridley, 2008
ROCK2Convergence and extensionMarlow et al., 2002Migration and invasionItoh et al., 1999
PAR1Convergence and extensionKusakabe and Nishida, 2004MigrationShi et al., 2004
PRKCIConvergence and extensionKusakabe and Nishida, 2004EMTGunaratne et al., 2013
CAP1Convergence and extensionSeifert et al., 2009MigrationYamazaki et al., 2009
EZREpibolyLink et al., 2006MigrationKhanna et al., 2004
EPCAMEpibolySlanchev et al., 2009Migration and invasionNi et al., 2012
ITGB1/ ITA5Mesodermal migrationSkalski et al., 1998Migration and invasionFelding-Habermann, 2003
FN1Convergence and extensionMarsden and DeSimone, 2003InvasionMalik et al., 2010
HAS2Dorsal migration of lateral cellsBakkers et al., 2004InvasionKim et al., 2004
MMP14Convergence and extensionCoyle et al., 2008InvasionPerentes et al., 2011
COX1EpibolyCha et al., 2006InvasionKundu and Fulton, 2002
PTGESConvergence and extensionSpeirs et al., 2010InvasionWang and Dubois, 2006
SLC39A6Anterior migrationYamashita et al., 2004EMTLue et al., 2011
GNA12 /13Convergence and extensionLin et al., 2005Migration and invasionYagi et al., 2011
OGTEpibolyWebster et al., 2009Migration and invasionLynch et al., 2012
CCN1Cell movementLatinkic et al., 2003Migration and invasionLin et al., 2012
TRPM7Convergence and extensionLiu et al., 2011MigrationMiddelbeek et al., 2012
MAPKAPK2EpibolyHolloway et al., 2009MigrationKumar et al., 2010
B4GALT1Convergence and extensionMachingo et al., 2006InvasionZhu et al., 2005
IER2Convergence and extensionHong et al., 2011MigrationNeeb et al., 2012
TIP1Convergence and extensionBesser et al., 2007Migration and invasionHan et al., 2012
PAK5Convergence and extensionFaure et al., 2005MigrationGong et al., 2009
MARCKSConvergence and extensionIioka et al., 2004Migration and invasionRombouts et al., 2013
Table 2
A list of the drugs that interfere with epiboly progression in zebrafish.

Related to Figure 1. A list of positive ‘hit’ drugs that interfered with epiboly progression. Gastrulation defects or status of each of the zebrafish embryos that were treated with either 10 or 50 μM concentrations are indicated.

Chemical nameChemical formulaEffect of 10 µMEffect of 50 µM
AcitretinC21H26O3DelayedDelayed
AdrenosteroneC19H24O3DelayedDelayed
AlbendazoleC12H15N3O2SSevere delayedSevere delayed
Alfadolone acetateC23H34O5DelayedDelayed
AlfaxaloneC21H32O3DelayedDelayed
AlprostadilC20H34O5DelayedDelayed
AltrenogestC21H26O2Slightly delayedDelayed
AmpiroxicamC20H21N3O7SNon-effectDelayed
Anethole-trithioneC10H8OS3DelayedDelayed
Antimycin AC28H40N2O9DelayedDelayed
AvobenzoneC20H22O3DelayedDelayed
BenzoxiquineC16H11NO2Non-effectDelayed
BosentanC27H29N5O6SDelayedDelayed
Butoconazole nitrateC19H18Cl3N3O3SDelayedToxic lethal
Camptothecine (S,+)C20H16N2O4Severe delayedSevere delayed
Carbenoxolone disodium saltC34H48Na2O7DelayedToxic lethal
CarmofurC11H16FN3O3Slightly delayedDelayed
CarprofenC15H12ClNO2Severe delayedToxic lethal
CefdinirC14H13N5O5S2DelayedDelayed
CelecoxibC17H14F3N3O2SDelayedDelayed
ChlorambucilC14H19Cl2NO2Slightly delayedDelayed
ChlorhexidineC22H30Cl2N10Non-effectToxic lethal
Ciclopirox ethanolamineC14H24N2O3DelayedSevere delayed
CinoxacinC12H10N2O5DelayedSevere delayed
ClofibrateC12H15ClO3Non-effectSevere delayed
ClopidogrelC16H16ClNO2SNon-effectDelayed
Clorgyline hydrochlorideC13H16Cl3NODelayedDelayed
ColchicineC22H25NO6Non-effectDelayed
Deptropine citrateC29H35NO8DelayedDelayed
Desipramine hydrochlorideC18H23ClN2DelayedDelayed
Diclofenac sodiumC14H10Cl2NNaO2DelayedSevere delayed
DicumarolC19H12O6DelayedSevere delayed
DiethylstilbestrolC18H20O2DelayedToxic lethal
Dimaprit dihydrochlorideC6H17Cl2N3SSlightly delayedDelayed
DisulfiramC10H20N2S4DelayedDelayed
Dopamine hydrochlorideC8H12ClNO2DelayedDelayed
Eburnamonine (-)C19H22N2ODelayedDelayed
Ethaverine hydrochlorideC24H30ClNO4DelayedDelayed
EthinylestradiolC20H24O2DelayedSevere delayed
Ethopropazine hydrochlorideC19H25ClN2SDelayedDelayed
EthoxyquinC14H19NONon-effectDelayed
ExemestaneC20H24O2Slightly delayedDelayed
EzetimibeC24H21F2NO3Slightly delayedDelayed
FenbendazoleC15H13N3O2SNon-effectDelayed
Fenoprofen calcium salt dihydrateC30H30CaO8Slightly delayedDelayed
FentiazacC17H12ClNO2SToxic lethalToxic lethal
FloxuridineC9H11FN2O5DelayedToxic lethal
Flunixin meglumineC21H28F3N3O7DelayedToxic lethal
FlutamideC11H11F3N2O3DelayedToxic lethal
Fluticasone propionateC25H31F3O5SNon-effectDelayed
FurosemideC12H11ClN2O5SDelayedDelayed
GatifloxacinC19H22FN3O4DelayedDelayed
GemcitabineC9H11F2N3O4DelayedDelayed
GemfibrozilC15H22O3DelayedToxic lethal
GestrinoneC21H24O2DelayedDelayed
HaloproginC9H4Cl3IODelayedToxic lethal
HexachloropheneC13H6Cl6O2DelayedSevere delayed
HexestrolC18H22O2Slightly delayedDelayed
IbudilastC14H18N2ONon-effectDelayed
Idazoxan hydrochlorideC11H13ClN2O2Slightly delayedDelayed
Idazoxan hydrochlorideC11H13ClN2O2Non-effectDelayed
IdebenoneC19H30O5Severe delayedToxic lethal
IndomethacinC19H16ClNO4Non-effectDelayed
IpriflavoneC18H16O3DelayedSevere delayed
IsotretinoinC20H28O2Non-effectSevere delayed
IsradipineC19H21N3O5Non-effectDelayed
LansoprazoleC16H14F3N3O2SSlightly delayedDelayed
LatanoprostC26H40O5Non-effectDelayed
LeflunomideC12H9F3N2O2DelayedSevere delayed
LetrozoleC17H11N5Non-effectDelayed
Lithocholic acidC24H40O3Non-effectDelayed
LodoxamideC11H6ClN3O6Non-effectDelayed
LofepramineC26H27ClN2ONon-effectDelayed
LoratadineC22H23ClN2O2DelayedDelayed
Loxapine succinateC22H24ClN3O5DelayedDelayed
MebendazoleC16H13N3O3Severe delayedSevere delayed
MebendazoleC22H26N2O2Non-effectDelayed
MeloxicamC14H13N3O4S2DelayedToxic lethal
MethiazoleC12H15N3O2SDelayedDelayed
MevastatinC23H34O5Non-effectDelayed
MK 801 hydrogen maleateC20H19NO4Slightly delayedDelayed
NabumetoneC15H16O2Non-effectSevere delayed
Naftopidil dihydrochlorideC24H30Cl2N2O3Slightly delayedDelayed
NandroloneC18H26O2DelayedDelayed
Naproxen sodium saltC14H13NaO3DelayedDelayed
NiclosamideC13H8Cl2N2O4DelayedDelayed
NifekalantC19H27N5O5DelayedDelayed
Niflumic acidC13H9F3N2O2DelayedDelayed
NimesulideC13H12N2O5SNon-effectDelayed
NisoldipineC20H24N2O6DelayedToxic lethal
NitazoxanideC12H9N3O5SSevere delayedSevere delayed
NorethindroneC20H26O2Non-effectDelayed
NorgestimateC23H31NO3Slightly delayedDelayed
OxfendazolC15H13N3O3SSlightly delayedDelayed
OxibendazolC12H15N3O3Severe delayedSevere delayed
OxymetholoneC21H32O3Slightly delayedDelayed
ParbendazoleC13H17N3O2Severe delayedSevere delayed
ParthenolideC15H20O3Non-effectDelayed
PenciclovirC10H15N5O3Non-effectDelayed
PentobarbitalC11H18N2O3Non-effectDelayed
Phenazopyridine hydrochlorideC11H12ClN5DelayedToxic lethal
PhenothiazineC12H9NSNon-effectDelayed
Phenoxybenzamine hydrochlorideC18H23Cl2NONon-effectDelayed
Pizotifen malateC23H27NO5SDelayedSevere delayed
Pramoxine hydrochlorideC17H28ClNO3Slightly delayedDelayed
Prilocaine hydrochlorideC13H21ClN2ONon-effectDelayed
PrimidoneC12H14N2O2Slightly delayedDelayed
RacecadotrilC21H23NO4SSlightly delayedDelayed
Riluzole hydrochlorideC8H6ClF3N2OSNon-effectDelayed
RitonavirC37H48N6O5S2Non-effectSevere delayed
S(-)Eticlopride hydrochlorideC17H26Cl2N2O3DelayedDelayed
SalmeterolC25H37NO4Non-effectDelayed
Streptomycin sulfateC42H84N14O36S3Non-effectDelayed
Sulconazole nitrateC18H16Cl3N3O3SDelayedDelayed
TegafurC8H9FN2O3DelayedDelayed
TelmisartanC33H30N4O2Severe delayedToxic lethal
TenatoprazoleC16H18N4O3SNon-effectDelayed
TerbinafineC21H25NNon-effectDelayed
ThimerosalC9H9HgNaO2SNon-effectDelayed
ThiorphanC12H15NO3SDelayedDelayed
TolcaponeC14H11NO5Severe delayedSevere delayed
TopotecanC23H23N3O5DelayedDelayed
Tracazolate hydrochlorideC16H25ClN4O2Severe delayedDelayed
TribenosideC29H34O6DelayedDelayed
TriclabendazoleC14H9Cl3N2OSDelayedDelayed
TriclosanC12H7Cl3O2DelayedSevere delayed
TrioxsalenC14H12O3DelayedDelayed
TroglitazoneC24H27NO5SSevere delayedToxic lethal
Valproic acidC8H16O2Non-effectDelayed
VoriconazoleC16H14F3N5ONon-effectDelayed
ZardaverineC12H10F2N2O3Slightly delayedDelayed
Zuclopenthixol dihydrochlorideC22H27Cl3N2OSDelayedDelayed
Table 3
Primary targets of the identified drugs.
The identified drugsPrimary targets of the identified drugs
HexachloropheneD-Lactate dehydrogenase (D-LDH), not expressed in mammalian cells
TroglitazoneAgonist for peroxisome proliferator-activated receptor α and γ (PPARα and -γ)
Pizotifen malate5-Hydroxytryptamine receptor 2C (HTR2C)
SalmeterolAdrenergic receptor beta 2 (ADRB2)
NitazoxanidePyruvate ferredoxin oxidoreductase (PFOR), not expressed in mammalian cells
Valproic acidHistone deacetylases (HDACs)
DicumarolNAD(P)H dehydrogenase quinone 1 (NQO1)
Loxapine succinateDopamine receptor D2 and D4 (DRD2 and DRD4)
AdrenosteroneHydroxysteroid (11-beta) dehydrogenase 1 (HSD11β1)
Riluzole hydrochlorideGlutamate R andvoltage-dependent Na+ channel
Naftopidil dihydrochloride5-Hydroxytryptamine receptor 1A (HTR1A) andα1-adrenergic receptor (AR)
S(-)Eticlopride hydrochlorideDopamine receptor D2 (DRD2)
RacecadotrilMembrane metallo-endopeptidase (MME)
IpriflavoneUnknown
FlurbiprofenCyclooxygenase 1 and 2 (Cox1 and -2)
ZardaverinePhosphodiesterase III/IV (PDE3/4)
LeflunomideDihydroorotate dehydrogenase (DHODH)
OlmesartanAngiotensin II receptor alpha
DisulfiramAldehyde dehydrogenase (ALDH)Dopamine β-hydroxylase (DBH)
Zuclopenthixol dihydrochlorideDopamine receptors D1 and D2 (DRD1 and -2)
Table 4
Effects of pharmacological inhibition of HTR2C on metastatic dissemination of MDA-MB-231 cells in zebrafish xenografted models.

Related to Figure 2D. The numbers and frequencies of the fish showing the dissemination patterns in vehicle- or pizotifen-treated group were indicated. The fish showed both patterns of dissemination were redundantly counted in this analysis.

Experiment_#1Experiment_#2Experiment_#3Average of experiments
 Drug: VehicleCell: MDA-MB-231Non-dissemination0% n1 = 0/170% n2 = 0/126.66% n3 = 1/152.22% ± 3.84%
Head58.82% n1 = 10/1791.66% n2 = 11/126.66% n3 = 1/1572.38% ± 17.15%
Trunk52.94% n1 = 9/178.33% n2 = 1/1220% n3 = 2/1527.09% ± 23.13%
End-tail100% n1 = 17/17100% n2 = 12/1286.66% n3 = 13/1595.55% ± 7.69%
 Drug: PizotifenCell: MDA-MB-231Non-dissemination55% n1 = 11/2031.57% n2 = 6/1945.45 % n3 = 10/2244.01% ± 11.77%
Head5% n1 = 1/2031.57% n2 = 6/1918.18% n3 = 4/2218.25% ± 13.28%
Trunk5% n1 = 1/2010.52% n2 = 2/194.45% n3 = 1/226.69% ± 3.32%
End-tail45% n1 = 9/2057.89% n2 = 11/1950% n3 = 11/2250.96% ± 6.50%
Table 5
Effects of pharmacological inhibition of HTR2C on metastatic dissemination of Mia-PaCa2 cells in zebrafish xenografted models.

Related to Figure 4. The numbers and frequencies of the fish showing the dissemination patterns in vehicle- or pizotifen-treated group were indicated. The fish showed both patterns of dissemination were redundantly counted in this analysis.

Experiment_#1Experiment_#2Average of experiments
 Drug: VehicleCell: MIA-PaCa2Non-dissemination17.64% n1 = 3/1716.66% n2 = 2/1217.15% + 0.69%
Head82.35% n1 = 14/1766.66% n2 = 8/1274.50% + 11.09%
Trunk29.41% n1 = 5/178.33% n2 = 1/1218.87% + 14.90%
End-tail70.58% n1 = 12/1783.33% n2 = 10/1776.96% + 9.01
 Drug: PizotifenCell: MIA-PaCa2Non-dissemination40% n1 = 4/1052.63% n2 = 10/1946.31% + 8.93%
Head20% n1 = 2/1010.52% n2 = 2/1915.26% + 6.69%
Trunk10% n1 = 1/105.26% n2 = 1/197.63% + 3.34%
End-tail40% n1 = 4/1042.05% n2 = 8/1941.4% + 1.48%
Table 6
Effects of genetic inhibition of HTR2C on metastatic dissemination of MDA-MB-231 cells in zebrafish xenografted models.

Related to Figure 2E. The numbers and frequencies of the fish showing the dissemination patterns in the zebrafish that were inoculated with either shLacZ or shHTR2C MDA-MB-231 cells were indicated. The fish showed both patterns of dissemination were redundantly counted in this analysis.

Experiment_#1Experiment_#2Average of experiments
 shLacZNon-dissemination0% n1 = 0/100% n2 = 0/100%
Head60% n1 = 6/10100% n2 = 10/1080%± 28.28%
Trunk30% n1 = 3/1010% n2 = 1/1020% ± 14.14%
End-tail80% n1 = 8/10100% n2 = 10/1090% ± 14.14
 shHTR2CNon-dissemination80% n1 = 12/1576.84% n2 = 14/1976.84 ± 4.46%
Head6.66% n1 = 1/1515.78% n2 = 3/1911.22% ± 6.45%
Trunk6.66% n1 = 1/155.26% n2 = 1/195.96% ± 0.99%
End-tail20% n1 = 3/1526.31% n2 = 5/1923.15% ± 4.46%
Table 7
Effects of HTR2C overexpression on metastatic dissemination of MCF7 cells in zebrafish xenografted models.

Related to Figure 4E. The numbers and frequencies of the fish showing the dissemination patterns in the zebrafish that were inoculated with MCF7 cells expressing either vector control (VC) or HTR2C were indicated. The fish showed both patterns of dissemination were redundantly counted in this analysis.

Experiment_#1Experiment_#2Average of experiments
VCNon-dissemination46.15% n1 = 6/1340% n2 = 4/1043.07% ± 4.35%
Head46.15% n1 = 6/1320% n2 = 2/1033.07% ± 18.49%
Trunk0% n1 = 0/130% n2 = 0/100%
End-tail53.84% n1 = 7/1360% n2 = 6/1056.92% ± 4.35%
 HTR2CNon-dissemination0% n1 = 0/140% n2 = 0/150%
Head100% n1 = 14/1493.33% n2 = 14/1596.66% ± 4.71%
Trunk64.28% n1 = 9/1473.33% n2 = 11/1568.80% ± 6.39%
End-tail85.71% n1 = 12/1493.33% n2 = 14/1589.52% ± 5.38%
Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Strain, strain background (Zebrafish)AB lineNational University of Singapore
Strain, strain background (Zebrafish)Tg (kdrl:eGFP) zebrafishProvided by Dr Stainier
Strain, strain background (Mus musculus)BALB/cCharles River Laboratories
Cell line (Homo sapiens)MDA-MB-231ATCCHTB-26
Cell line (Homo sapiens)MCF7ATCCHTB-22
Cell line (Homo sapiens)MDA-MB-435ATCCHTB-129
Cell line (Homo sapiens)MIA-PaCa2ATCCCRM-CRL-1420
Cell line (Homo sapiens)PC3ATCCCRL-3471
Cell line (Homo sapiens)SW620ATCCCCL-227
Cell line (Homo sapiens)PC9RIKEN BRCRCB0446
Cell line (Homo sapiens)HaCaTCLI300493
Cell line (BALB/c Mus)4T1-12BProvided from Dr Gary Sahagian
AntibodyPRMT1 (A33)(Rabbit polyclonal)Cell Signaling TechnologyCat#_2449WB (1:1000)
AntibodyCYP11A1 (D8F4F)(Rabbit polyclonal)Cell Signaling TechnologyCat#_14217WB (1:1000)
AntibodyE-cadherin (4A2)(Mouse monoclonal)Cell Signaling TechnologyCat#_14472WB (1:1000)IF (1:100)
AntibodyEpCAM (VU1D9)(Mouse monoclonal)Cell Signaling TechnologyCat#_2929WB (1:1000)IF (1:100)
AntibodyVimentin (D21H3)(Rabbit polyclonal)Cell Signaling TechnologyCat#_5741WB (1:1000)IF (1:100)
AntibodyN-cadherin (D4R1H)(Rabbit polyclonal)Cell Signaling TechnologyCat#_13116WB (1:1000)IF (1:100)
AntibodyZEB1 (D80D3)(Rabbit polyclonal)Cell Signaling TechnologyCat#_3396WB (1:1000)
AntibodyHistone H3 (D1H2)(Rabbit polyclonal)Cell Signaling TechnologyCat#_4499WB (1:1000)
Antibodyβ-Tubulin (9F3)(Rabbit polyclonal)Cell Signaling TechnologyCat#_2128WB (1:1000)
AntibodyGAPDH (14C10)(Rabbit polyclonal)Cell Signaling TechnologyCat#_2118WB (1:1000)
AntibodyHTR2C (ab133570)(Rabbit polyclonal)AbcamCat#_ab133570WB (1:1000)
AntibodyDRD2 (ab85367)(Rabbit polyclonal)AbcamCat#_ab85367WB (1:1000)
AntibodyPhospho-GSK3β (Ser9) (F-2)(Mouse monoclonal)Santa Cruz BiotechnologyCat#_sc-373800WB (1:1000)
AntibodyGSK3β (1F7)(Mouse monoclonal)Santa Cruz BiotechnologyCat#_sc-53931WB (1:1000)
AntibodyKRT18 (DC-10)(Mouse monoclonal)Santa Cruz BiotechnologyCat#_sc-6259WB (1:1000)
AntibodyKRT19 (A53-B/A2)(Mouse monoclonal)Santa Cruz BiotechnologyCat#_sc-6278WB (1:1000)
AntibodyMMP1 (3B6)(Mouse monoclonal)Santa Cruz BiotechnologyCat#_sc-21731WB (1:1000)
AntibodyMMP2 (8B4)(Mouse monoclonal)Santa Cruz BiotechnologyCat#_sc-13595WB (1:1000)
AntibodyS100A4 (A-7)(Mouse monoclonal)Santa Cruz BiotechnologyCat#_sc-377059WB (1:1000)
AntibodyLuciferase (C-12)(Mouse monoclonal)Santa Cruz BiotechnologyCat#_sc-74548WB (1:1000)
Antibodyki67 (ki-67)(Mouse monoclonal)Santa Cruz BiotechnologyCat#_sc-23900WB (1:1000)
Antibodyβ-Catenin (E-5)(Mouse monoclonal)Santa Cruz BiotechnologyCat#_sc-7963WB (1:1000)IF (1:100)
AntibodyFITC-conjugated E-cadherin antibody (67A4)BiolegendCat#_324104FACS (1:100)
AntibodyAnti-mouse anti-rabbit immunoglobulin G (IgG) antibodies conjugated to Alexa Fluor 488Life TechnologiesA-11029IF (1:100)
AntibodyAnti-goat anti-rabbit immunoglobulin G (IgG) antibodies conjugated to Alexa Fluor 488Life TechnologiesA-11034IF (1:100)
Recombinant DNA reagentpLVX-shRNA1ClontechCat#_ 632,177
Recombinant DNA reagentpCDH-CMV-MCS-EF1α-HygroSystem BiosciencesCat#_CD515B-1Gene expression vector
Recombinant DNA reagentpMDLg/pRREAddgeneAddgene Plasmid #12251 RRID:Addgene_12251Lentivirus packaging vector
Recombinant DNA reagentpRSV-revAddgeneAddgene Plasmid #12253 RRID:Addgene_12253Lentivirus packaging vector
Recombinant DNA reagentpMD2.GAddgeneAddgene Plasmid #12259 RRID:Addgene_12259Lentivirus packaging vector
Recombinant DNA reagentProviding pCMV-h5TH2C-VSVProvided from Dr Herrick
Chemical compound, drugFDA-approved chemical librariesPrestwick Chemical
Chemical compound, drugPizotifenSanta Cruz BiotechnologyCat#_sc-201143
Chemical compound, drugS(-)Eticlopride hydrochlorideSanta Cruz BiotechnologyCat#_E101
Software, algorithmGraphPad Prism7GraphPad Software IncRRID:SCR_002798Data analysis
Software, algorithmFlowJoBD BiosciencesRRID:SCR_008520FACS data analysis

Additional files

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Source data 1

GSEA analysis of zebrafish embryos at either 50%-epiboly, shield or 75%-epiboly stage.

https://cdn.elifesciences.org/articles/70151/elife-70151-data1-v3.xlsx
Source data 2

Enriched pathways of zebrafish embryos at either 50%-epiboly, shield or 75%-epiboly stage.

https://cdn.elifesciences.org/articles/70151/elife-70151-data2-v3.xlsx
Source data 3

Raw data of western-blotting analysis.

https://cdn.elifesciences.org/articles/70151/elife-70151-data3-v3.zip
Source data 4

Raw data of western-blotting analysis with legends.

https://cdn.elifesciences.org/articles/70151/elife-70151-data4-v3.docx

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  1. Joji Nakayama
  2. Lora Tan
  3. Yan Li
  4. Boon Cher Goh
  5. Shu Wang
  6. Hideki Makinoshima
  7. Zhiyuan Gong
(2021)
A zebrafish embryo screen utilizing gastrulation identifies the HTR2C inhibitor pizotifen as a suppressor of EMT-mediated metastasis
eLife 10:e70151.
https://doi.org/10.7554/eLife.70151