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

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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.

Genes	Gastrulation defects	Ref	Effects in metastasis	Ref
BMP	Convergence and extension	Kondo, 2007	EMT	Katsuno et al., 2008
WNT	Convergence and extension	Tada and Smith, 2000	Migration and invasion	Vincan and Barker, 2008
FGF	Convergence and extension	Yang et al., 2002	Invision	Nomura et al., 2008
EGF	Epiboly	Song et al., 2013	Migration	Lu et al., 2001
PDGF	Convergence and extension	Damm and Winklbauer, 2011	EMT	Jechlinger et al., 2006
CXCL12	Migration of endodermal cells	Mizoguchi et al., 2008	Migration and invasion	Shen et al., 2013
CXCR4	Migration of endodermal cells	Mizoguchi et al., 2008	Migration and invasion	Shen et al., 2013
PIK3CA	Convergence and extension	Montero et al., 2003	Migration and invasion	Wander et al., 2013
YES	Epiboly	Tsai et al., 2005	Migration	Barraclough et al., 2007
FYN	Epiboly	Sharma et al., 2005	Migration and invasion	Yadav and Denning, 2011
MAPK1	Epiboly	Krens et al., 2008	Migration	Radtke et al., 2013
SHP2	Convergence and extension	Jopling et al., 2007	Migration	Wang et al., 2005
SNAI1	Convergence and extension	Ip and Gridley, 2002	EMT	Batlle et al., 2000
SNAI2	Mesoderm and neural crest formation	Shi et al., 2011	EMT	Medici et al., 2008
TWIST1	Mesoderm formation	Castanon and Baylies, 2002	EMT	Yang et al., 2004
TBXT	Convergence and extension	Tada and Smith, 2000	EMT	Fernando et al., 2010
ZEB1	Epiboly	Vannier et al., 2013	EMT	Spaderna et al., 2008
GSC	Mesodermal patterning	Sander et al., 2007	EMT	Hartwell et al., 2006
FOXC2	Unclear, defects in gastrulation	Wilm et al., 2004	EMT	Mani et al., 2007
STAT3	Convergence and extension	Miyagi et al., 2004	Migration	Abdulghani et al., 2008
POU5F1	Epiboly	Lachnit et al., 2008	EMT	Dai et al., 2013
EZH2	Unclear, defects in gastrulation	O’Carroll et al., 2001	Invasion	Ren et al., 2012
EHMT2	Defects in neurogenesis	Lin et al., 2005	Migration and invasion	Chen et al., 2010
BMI1	Defects in skeleton formation	van der Lugt et al., 1994	EMT	Guo et al., 2011
RHOA	Convergence and extension	Zhu et al., 2006	Migration and invasion	Yoshioka et al., 1999
CDC42	Convergence and extension	Choi and Han, 2002	Migration and invasion	Reymond et al., 2012
RAC1	Convergence and extension	Habas et al., 2003	Migration and invasion	Vega and Ridley, 2008
ROCK2	Convergence and extension	Marlow et al., 2002	Migration and invasion	Itoh et al., 1999
PAR1	Convergence and extension	Kusakabe and Nishida, 2004	Migration	Shi et al., 2004
PRKCI	Convergence and extension	Kusakabe and Nishida, 2004	EMT	Gunaratne et al., 2013
CAP1	Convergence and extension	Seifert et al., 2009	Migration	Yamazaki et al., 2009
EZR	Epiboly	Link et al., 2006	Migration	Khanna et al., 2004
EPCAM	Epiboly	Slanchev et al., 2009	Migration and invasion	Ni et al., 2012
ITGB1/ ITA5	Mesodermal migration	Skalski et al., 1998	Migration and invasion	Felding-Habermann, 2003
FN1	Convergence and extension	Marsden and DeSimone, 2003	Invasion	Malik et al., 2010
HAS2	Dorsal migration of lateral cells	Bakkers et al., 2004	Invasion	Kim et al., 2004
MMP14	Convergence and extension	Coyle et al., 2008	Invasion	Perentes et al., 2011
COX1	Epiboly	Cha et al., 2006	Invasion	Kundu and Fulton, 2002
PTGES	Convergence and extension	Speirs et al., 2010	Invasion	Wang and Dubois, 2006
SLC39A6	Anterior migration	Yamashita et al., 2004	EMT	Lue et al., 2011
GNA12 /13	Convergence and extension	Lin et al., 2005	Migration and invasion	Yagi et al., 2011
OGT	Epiboly	Webster et al., 2009	Migration and invasion	Lynch et al., 2012
CCN1	Cell movement	Latinkic et al., 2003	Migration and invasion	Lin et al., 2012
TRPM7	Convergence and extension	Liu et al., 2011	Migration	Middelbeek et al., 2012
MAPKAPK2	Epiboly	Holloway et al., 2009	Migration	Kumar et al., 2010
B4GALT1	Convergence and extension	Machingo et al., 2006	Invasion	Zhu et al., 2005
IER2	Convergence and extension	Hong et al., 2011	Migration	Neeb et al., 2012
TIP1	Convergence and extension	Besser et al., 2007	Migration and invasion	Han et al., 2012
PAK5	Convergence and extension	Faure et al., 2005	Migration	Gong et al., 2009
MARCKS	Convergence and extension	Iioka et al., 2004	Migration and invasion	Rombouts 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 name	Chemical formula	Effect of 10 µM	Effect of 50 µM
Acitretin	C₂₁H₂₆O₃	Delayed	Delayed
Adrenosterone	C₁₉H₂₄O₃	Delayed	Delayed
Albendazole	C₁₂H₁₅N₃O₂S	Severe delayed	Severe delayed
Alfadolone acetate	C₂₃H₃₄O₅	Delayed	Delayed
Alfaxalone	C₂₁H₃₂O₃	Delayed	Delayed
Alprostadil	C₂₀H₃₄O₅	Delayed	Delayed
Altrenogest	C₂₁H₂₆O₂	Slightly delayed	Delayed
Ampiroxicam	C₂₀H₂₁N₃O₇S	Non-effect	Delayed
Anethole-trithione	C₁₀H_8OS₃	Delayed	Delayed
Antimycin A	C₂₈H₄₀N₂O₉	Delayed	Delayed
Avobenzone	C₂₀H₂₂O₃	Delayed	Delayed
Benzoxiquine	C₁₆H₁₁NO₂	Non-effect	Delayed
Bosentan	C₂₇H₂₉N₅O₆S	Delayed	Delayed
Butoconazole nitrate	C₁₉H₁₈C_l3N₃O₃S	Delayed	Toxic lethal
Camptothecine (S,+)	C₂₀H₁₆N₂O₄	Severe delayed	Severe delayed
Carbenoxolone disodium salt	C₃₄H₄₈Na₂O₇	Delayed	Toxic lethal
Carmofur	C₁₁H₁₆FN₃O₃	Slightly delayed	Delayed
Carprofen	C₁₅H₁₂ClNO₂	Severe delayed	Toxic lethal
Cefdinir	C₁₄H₁₃N₅O₅S₂	Delayed	Delayed
Celecoxib	C₁₇H₁₄F₃N₃O₂S	Delayed	Delayed
Chlorambucil	C₁₄H₁₉C_l2NO₂	Slightly delayed	Delayed
Chlorhexidine	C₂₂H₃₀C_l2N₁₀	Non-effect	Toxic lethal
Ciclopirox ethanolamine	C₁₄H₂₄N₂O₃	Delayed	Severe delayed
Cinoxacin	C₁₂H₁₀N₂O₅	Delayed	Severe delayed
Clofibrate	C₁₂H₁₅ClO₃	Non-effect	Severe delayed
Clopidogrel	C₁₆H₁₆ClNO₂S	Non-effect	Delayed
Clorgyline hydrochloride	C₁₃H₁₆Cl₃NO	Delayed	Delayed
Colchicine	C₂₂H₂₅NO₆	Non-effect	Delayed
Deptropine citrate	C₂₉H₃₅NO₈	Delayed	Delayed
Desipramine hydrochloride	C₁₈H₂₃ClN₂	Delayed	Delayed
Diclofenac sodium	C₁₄H₁₀Cl₂NNaO₂	Delayed	Severe delayed
Dicumarol	C₁₉H₁₂O₆	Delayed	Severe delayed
Diethylstilbestrol	C₁₈H₂₀O₂	Delayed	Toxic lethal
Dimaprit dihydrochloride	C₆H₁₇Cl₂N₃S	Slightly delayed	Delayed
Disulfiram	C₁₀H₂₀N₂S₄	Delayed	Delayed
Dopamine hydrochloride	C₈H₁₂ClNO₂	Delayed	Delayed
Eburnamonine (-)	C₁₉H₂₂N₂O	Delayed	Delayed
Ethaverine hydrochloride	C₂₄H₃₀ClNO₄	Delayed	Delayed
Ethinylestradiol	C₂₀H₂₄O₂	Delayed	Severe delayed
Ethopropazine hydrochloride	C₁₉H₂₅ClN₂S	Delayed	Delayed
Ethoxyquin	C₁₄H₁₉NO	Non-effect	Delayed
Exemestane	C₂₀H₂₄O₂	Slightly delayed	Delayed
Ezetimibe	C₂₄H₂₁F₂NO₃	Slightly delayed	Delayed
Fenbendazole	C₁₅H₁₃N₃O₂S	Non-effect	Delayed
Fenoprofen calcium salt dihydrate	C₃₀H₃₀CaO₈	Slightly delayed	Delayed
Fentiazac	C₁₇H₁₂ClNO₂S	Toxic lethal	Toxic lethal
Floxuridine	C₉H₁₁FN₂O₅	Delayed	Toxic lethal
Flunixin meglumine	C₂₁H₂₈F₃N₃O₇	Delayed	Toxic lethal
Flutamide	C₁₁H₁₁F₃N₂O₃	Delayed	Toxic lethal
Fluticasone propionate	C₂₅H₃₁F₃O₅S	Non-effect	Delayed
Furosemide	C₁₂H₁₁ClN₂O₅S	Delayed	Delayed
Gatifloxacin	C₁₉H₂₂FN₃O₄	Delayed	Delayed
Gemcitabine	C₉H₁₁F₂N₃O₄	Delayed	Delayed
Gemfibrozil	C₁₅H₂₂O₃	Delayed	Toxic lethal
Gestrinone	C₂₁H₂₄O₂	Delayed	Delayed
Haloprogin	C₉H₄Cl₃IO	Delayed	Toxic lethal
Hexachlorophene	C₁₃H₆Cl₆O₂	Delayed	Severe delayed
Hexestrol	C₁₈H₂₂O₂	Slightly delayed	Delayed
Ibudilast	C₁₄H₁₈N₂O	Non-effect	Delayed
Idazoxan hydrochloride	C₁₁H₁₃ClN₂O₂	Slightly delayed	Delayed
Idazoxan hydrochloride	C₁₁H₁₃ClN₂O₂	Non-effect	Delayed
Idebenone	C₁₉H₃₀O₅	Severe delayed	Toxic lethal
Indomethacin	C₁₉H₁₆ClNO₄	Non-effect	Delayed
Ipriflavone	C₁₈H₁₆O₃	Delayed	Severe delayed
Isotretinoin	C₂₀H₂₈O₂	Non-effect	Severe delayed
Isradipine	C₁₉H₂₁N₃O₅	Non-effect	Delayed
Lansoprazole	C₁₆H₁₄F₃N₃O₂S	Slightly delayed	Delayed
Latanoprost	C₂₆H₄₀O₅	Non-effect	Delayed
Leflunomide	C₁₂H₉F₃N₂O₂	Delayed	Severe delayed
Letrozole	C₁₇H₁₁N₅	Non-effect	Delayed
Lithocholic acid	C₂₄H₄₀O₃	Non-effect	Delayed
Lodoxamide	C₁₁H₆ClN₃O₆	Non-effect	Delayed
Lofepramine	C₂₆H₂₇ClN₂O	Non-effect	Delayed
Loratadine	C₂₂H₂₃ClN₂O₂	Delayed	Delayed
Loxapine succinate	C₂₂H₂₄ClN₃O₅	Delayed	Delayed
Mebendazole	C₁₆H₁₃N₃O₃	Severe delayed	Severe delayed
Mebendazole	C₂₂H₂₆N₂O₂	Non-effect	Delayed
Meloxicam	C₁₄H₁₃N₃O₄S₂	Delayed	Toxic lethal
Methiazole	C₁₂H₁₅N₃O₂S	Delayed	Delayed
Mevastatin	C₂₃H₃₄O₅	Non-effect	Delayed
MK 801 hydrogen maleate	C₂₀H₁₉NO₄	Slightly delayed	Delayed
Nabumetone	C₁₅H₁₆O₂	Non-effect	Severe delayed
Naftopidil dihydrochloride	C₂₄H₃₀Cl₂N₂O₃	Slightly delayed	Delayed
Nandrolone	C₁₈H₂₆O₂	Delayed	Delayed
Naproxen sodium salt	C₁₄H₁₃NaO₃	Delayed	Delayed
Niclosamide	C₁₃H₈Cl₂N₂O₄	Delayed	Delayed
Nifekalant	C₁₉H₂₇N₅O₅	Delayed	Delayed
Niflumic acid	C₁₃H₉F₃N₂O₂	Delayed	Delayed
Nimesulide	C₁₃H₁₂N₂O₅S	Non-effect	Delayed
Nisoldipine	C₂₀H₂₄N₂O₆	Delayed	Toxic lethal
Nitazoxanide	C₁₂H₉N₃O₅S	Severe delayed	Severe delayed
Norethindrone	C₂₀H₂₆O₂	Non-effect	Delayed
Norgestimate	C₂₃H₃₁NO₃	Slightly delayed	Delayed
Oxfendazol	C₁₅H₁₃N₃O₃S	Slightly delayed	Delayed
Oxibendazol	C₁₂H₁₅N₃O₃	Severe delayed	Severe delayed
Oxymetholone	C₂₁H₃₂O₃	Slightly delayed	Delayed
Parbendazole	C₁₃H₁₇N₃O₂	Severe delayed	Severe delayed
Parthenolide	C₁₅H₂₀O₃	Non-effect	Delayed
Penciclovir	C₁₀H₁₅N₅O₃	Non-effect	Delayed
Pentobarbital	C₁₁H₁₈N₂O₃	Non-effect	Delayed
Phenazopyridine hydrochloride	C₁₁H₁₂ClN₅	Delayed	Toxic lethal
Phenothiazine	C₁₂H₉NS	Non-effect	Delayed
Phenoxybenzamine hydrochloride	C₁₈H₂₃Cl₂NO	Non-effect	Delayed
Pizotifen malate	C₂₃H₂₇NO₅S	Delayed	Severe delayed
Pramoxine hydrochloride	C₁₇H₂₈ClNO₃	Slightly delayed	Delayed
Prilocaine hydrochloride	C₁₃H₂₁ClN₂O	Non-effect	Delayed
Primidone	C₁₂H₁₄N₂O₂	Slightly delayed	Delayed
Racecadotril	C₂₁H₂₃NO₄S	Slightly delayed	Delayed
Riluzole hydrochloride	C₈H₆ClF₃N₂OS	Non-effect	Delayed
Ritonavir	C₃₇H₄₈N₆O₅S₂	Non-effect	Severe delayed
S(-)Eticlopride hydrochloride	C₁₇H₂₆Cl₂N₂O₃	Delayed	Delayed
Salmeterol	C₂₅H₃₇NO₄	Non-effect	Delayed
Streptomycin sulfate	C₄₂H₈₄N₁₄O₃₆S₃	Non-effect	Delayed
Sulconazole nitrate	C₁₈H₁₆C_l3N₃O₃S	Delayed	Delayed
Tegafur	C₈H₉FN₂O₃	Delayed	Delayed
Telmisartan	C₃₃H₃₀N₄O₂	Severe delayed	Toxic lethal
Tenatoprazole	C₁₆H₁₈N₄O₃S	Non-effect	Delayed
Terbinafine	C₂₁H₂₅N	Non-effect	Delayed
Thimerosal	C₉H₉HgNaO₂S	Non-effect	Delayed
Thiorphan	C₁₂H₁₅NO₃S	Delayed	Delayed
Tolcapone	C₁₄H₁₁NO₅	Severe delayed	Severe delayed
Topotecan	C₂₃H₂₃N₃O₅	Delayed	Delayed
Tracazolate hydrochloride	C₁₆H₂₅ClN₄O₂	Severe delayed	Delayed
Tribenoside	C₂₉H₃₄O₆	Delayed	Delayed
Triclabendazole	C₁₄H₉Cl₃N₂OS	Delayed	Delayed
Triclosan	C₁₂H₇Cl₃O₂	Delayed	Severe delayed
Trioxsalen	C₁₄H₁₂O₃	Delayed	Delayed
Troglitazone	C₂₄H₂₇NO₅S	Severe delayed	Toxic lethal
Valproic acid	C₈H₁₆O₂	Non-effect	Delayed
Voriconazole	C₁₆H₁₄F₃N₅O	Non-effect	Delayed
Zardaverine	C₁₂H₁₀F₂N₂O₃	Slightly delayed	Delayed
Zuclopenthixol dihydrochloride	C₂₂H₂₇Cl₃N₂OS	Delayed	Delayed

Table 3

Primary targets of the identified drugs.

The identified drugs	Primary targets of the identified drugs
Hexachlorophene	D-Lactate dehydrogenase (D-LDH), not expressed in mammalian cells
Troglitazone	Agonist for peroxisome proliferator-activated receptor α and γ (PPARα and -γ)
Pizotifen malate	5-Hydroxytryptamine receptor 2C (HTR2C)
Salmeterol	Adrenergic receptor beta 2 (ADRB2)
Nitazoxanide	Pyruvate ferredoxin oxidoreductase (PFOR), not expressed in mammalian cells
Valproic acid	Histone deacetylases (HDACs)
Dicumarol	NAD(P)H dehydrogenase quinone 1 (NQO1)
Loxapine succinate	Dopamine receptor D2 and D4 (DRD2 and DRD4)
Adrenosterone	Hydroxysteroid (11-beta) dehydrogenase 1 (HSD11β1)
Riluzole hydrochloride	Glutamate R andvoltage-dependent Na+ channel
Naftopidil dihydrochloride	5-Hydroxytryptamine receptor 1A (HTR1A) andα1-adrenergic receptor (AR)
S(-)Eticlopride hydrochloride	Dopamine receptor D2 (DRD2)
Racecadotril	Membrane metallo-endopeptidase (MME)
Ipriflavone	Unknown
Flurbiprofen	Cyclooxygenase 1 and 2 (Cox1 and -2)
Zardaverine	Phosphodiesterase III/IV (PDE3/4)
Leflunomide	Dihydroorotate dehydrogenase (DHODH)
Olmesartan	Angiotensin II receptor alpha
Disulfiram	Aldehyde dehydrogenase (ALDH)Dopamine β-hydroxylase (DBH)
Zuclopenthixol dihydrochloride	Dopamine 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_#1	Experiment_#2	Experiment_#3	Average of experiments
Drug: VehicleCell: MDA-MB-231	Non-dissemination	0% n1 = 0/17	0% n2 = 0/12	6.66% n3 = 1/15	2.22% ± 3.84%
	Head	58.82% n1 = 10/17	91.66% n2 = 11/12	6.66% n3 = 1/15	72.38% ± 17.15%
	Trunk	52.94% n1 = 9/17	8.33% n2 = 1/12	20% n3 = 2/15	27.09% ± 23.13%
	End-tail	100% n1 = 17/17	100% n2 = 12/12	86.66% n3 = 13/15	95.55% ± 7.69%
Drug: PizotifenCell: MDA-MB-231	Non-dissemination	55% n1 = 11/20	31.57% n2 = 6/19	45.45 % n3 = 10/22	44.01% ± 11.77%
	Head	5% n1 = 1/20	31.57% n2 = 6/19	18.18% n3 = 4/22	18.25% ± 13.28%
	Trunk	5% n1 = 1/20	10.52% n2 = 2/19	4.45% n3 = 1/22	6.69% ± 3.32%
	End-tail	45% n1 = 9/20	57.89% n2 = 11/19	50% n3 = 11/22	50.96% ± 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_#1	Experiment_#2	Average of experiments
Drug: VehicleCell: MIA-PaCa2	Non-dissemination	17.64% n1 = 3/17	16.66% n2 = 2/12	17.15% + 0.69%
	Head	82.35% n1 = 14/17	66.66% n2 = 8/12	74.50% + 11.09%
	Trunk	29.41% n1 = 5/17	8.33% n2 = 1/12	18.87% + 14.90%
	End-tail	70.58% n1 = 12/17	83.33% n2 = 10/17	76.96% + 9.01
Drug: PizotifenCell: MIA-PaCa2	Non-dissemination	40% n1 = 4/10	52.63% n2 = 10/19	46.31% + 8.93%
	Head	20% n1 = 2/10	10.52% n2 = 2/19	15.26% + 6.69%
	Trunk	10% n1 = 1/10	5.26% n2 = 1/19	7.63% + 3.34%
	End-tail	40% n1 = 4/10	42.05% n2 = 8/19	41.4% + 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_#1	Experiment_#2	Average of experiments
shLacZ	Non-dissemination	0% n1 = 0/10	0% n2 = 0/10	0%
	Head	60% n1 = 6/10	100% n2 = 10/10	80%± 28.28%
	Trunk	30% n1 = 3/10	10% n2 = 1/10	20% ± 14.14%
	End-tail	80% n1 = 8/10	100% n2 = 10/10	90% ± 14.14
shHTR2C	Non-dissemination	80% n1 = 12/15	76.84% n2 = 14/19	76.84 ± 4.46%
	Head	6.66% n1 = 1/15	15.78% n2 = 3/19	11.22% ± 6.45%
	Trunk	6.66% n1 = 1/15	5.26% n2 = 1/19	5.96% ± 0.99%
	End-tail	20% n1 = 3/15	26.31% n2 = 5/19	23.15% ± 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_#1	Experiment_#2	Average of experiments
VC	Non-dissemination	46.15% n1 = 6/13	40% n2 = 4/10	43.07% ± 4.35%
	Head	46.15% n1 = 6/13	20% n2 = 2/10	33.07% ± 18.49%
	Trunk	0% n1 = 0/13	0% n2 = 0/10	0%
	End-tail	53.84% n1 = 7/13	60% n2 = 6/10	56.92% ± 4.35%
HTR2C	Non-dissemination	0% n1 = 0/14	0% n2 = 0/15	0%
	Head	100% n1 = 14/14	93.33% n2 = 14/15	96.66% ± 4.71%
	Trunk	64.28% n1 = 9/14	73.33% n2 = 11/15	68.80% ± 6.39%
	End-tail	85.71% n1 = 12/14	93.33% n2 = 14/15	89.52% ± 5.38%

Key resources table

Reagent type (species) or resource	Designation	Source or reference	Identifiers	Additional information
Strain, strain background (Zebrafish)	AB line	National University of Singapore
Strain, strain background (Zebrafish)	Tg (kdrl:eGFP) zebrafish	Provided by Dr Stainier
Strain, strain background (Mus musculus)	BALB/c	Charles River Laboratories
Cell line (Homo sapiens)	MDA-MB-231	ATCC	HTB-26
Cell line (Homo sapiens)	MCF7	ATCC	HTB-22
Cell line (Homo sapiens)	MDA-MB-435	ATCC	HTB-129
Cell line (Homo sapiens)	MIA-PaCa2	ATCC	CRM-CRL-1420
Cell line (Homo sapiens)	PC3	ATCC	CRL-3471
Cell line (Homo sapiens)	SW620	ATCC	CCL-227
Cell line (Homo sapiens)	PC9	RIKEN BRC	RCB0446
Cell line (Homo sapiens)	HaCaT	CLI	300493
Cell line (BALB/c Mus)	4T1-12B	Provided from Dr Gary Sahagian
Antibody	PRMT1 (A33)(Rabbit polyclonal)	Cell Signaling Technology	Cat#_2449	WB (1:1000)
Antibody	CYP11A1 (D8F4F)(Rabbit polyclonal)	Cell Signaling Technology	Cat#_14217	WB (1:1000)
Antibody	E-cadherin (4A2)(Mouse monoclonal)	Cell Signaling Technology	Cat#_14472	WB (1:1000)IF (1:100)
Antibody	EpCAM (VU1D9)(Mouse monoclonal)	Cell Signaling Technology	Cat#_2929	WB (1:1000)IF (1:100)
Antibody	Vimentin (D21H3)(Rabbit polyclonal)	Cell Signaling Technology	Cat#_5741	WB (1:1000)IF (1:100)
Antibody	N-cadherin (D4R1H)(Rabbit polyclonal)	Cell Signaling Technology	Cat#_13116	WB (1:1000)IF (1:100)
Antibody	ZEB1 (D80D3)(Rabbit polyclonal)	Cell Signaling Technology	Cat#_3396	WB (1:1000)
Antibody	Histone H3 (D1H2)(Rabbit polyclonal)	Cell Signaling Technology	Cat#_4499	WB (1:1000)
Antibody	β-Tubulin (9F3)(Rabbit polyclonal)	Cell Signaling Technology	Cat#_2128	WB (1:1000)
Antibody	GAPDH (14C10)(Rabbit polyclonal)	Cell Signaling Technology	Cat#_2118	WB (1:1000)
Antibody	HTR2C (ab133570)(Rabbit polyclonal)	Abcam	Cat#_ab133570	WB (1:1000)
Antibody	DRD2 (ab85367)(Rabbit polyclonal)	Abcam	Cat#_ab85367	WB (1:1000)
Antibody	Phospho-GSK3β (Ser9) (F-2)(Mouse monoclonal)	Santa Cruz Biotechnology	Cat#_sc-373800	WB (1:1000)
Antibody	GSK3β (1F7)(Mouse monoclonal)	Santa Cruz Biotechnology	Cat#_sc-53931	WB (1:1000)
Antibody	KRT18 (DC-10)(Mouse monoclonal)	Santa Cruz Biotechnology	Cat#_sc-6259	WB (1:1000)
Antibody	KRT19 (A53-B/A2)(Mouse monoclonal)	Santa Cruz Biotechnology	Cat#_sc-6278	WB (1:1000)
Antibody	MMP1 (3B6)(Mouse monoclonal)	Santa Cruz Biotechnology	Cat#_sc-21731	WB (1:1000)
Antibody	MMP2 (8B4)(Mouse monoclonal)	Santa Cruz Biotechnology	Cat#_sc-13595	WB (1:1000)
Antibody	S100A4 (A-7)(Mouse monoclonal)	Santa Cruz Biotechnology	Cat#_sc-377059	WB (1:1000)
Antibody	Luciferase (C-12)(Mouse monoclonal)	Santa Cruz Biotechnology	Cat#_sc-74548	WB (1:1000)
Antibody	ki67 (ki-67)(Mouse monoclonal)	Santa Cruz Biotechnology	Cat#_sc-23900	WB (1:1000)
Antibody	β-Catenin (E-5)(Mouse monoclonal)	Santa Cruz Biotechnology	Cat#_sc-7963	WB (1:1000)IF (1:100)
Antibody	FITC-conjugated E-cadherin antibody (67A4)	Biolegend	Cat#_324104	FACS (1:100)
Antibody	Anti-mouse anti-rabbit immunoglobulin G (IgG) antibodies conjugated to Alexa Fluor 488	Life Technologies	A-11029	IF (1:100)
Antibody	Anti-goat anti-rabbit immunoglobulin G (IgG) antibodies conjugated to Alexa Fluor 488	Life Technologies	A-11034	IF (1:100)
Recombinant DNA reagent	pLVX-shRNA1	Clontech	Cat#_ 632,177
Recombinant DNA reagent	pCDH-CMV-MCS-EF1α-Hygro	System Biosciences	Cat#_CD515B-1	Gene expression vector
Recombinant DNA reagent	pMDLg/pRRE	Addgene	Addgene Plasmid #12251 RRID:Addgene_12251	Lentivirus packaging vector
Recombinant DNA reagent	pRSV-rev	Addgene	Addgene Plasmid #12253 RRID:Addgene_12253	Lentivirus packaging vector
Recombinant DNA reagent	pMD2.G	Addgene	Addgene Plasmid #12259 RRID:Addgene_12259	Lentivirus packaging vector
Recombinant DNA reagent	Providing pCMV-h5TH2C-VSV	Provided from Dr Herrick
Chemical compound, drug	FDA-approved chemical libraries	Prestwick Chemical
Chemical compound, drug	Pizotifen	Santa Cruz Biotechnology	Cat#_sc-201143
Chemical compound, drug	S(-)Eticlopride hydrochloride	Santa Cruz Biotechnology	Cat#_E101
Software, algorithm	GraphPad Prism7	GraphPad Software Inc	RRID:SCR_002798	Data analysis
Software, algorithm	FlowJo	BD Biosciences	RRID:SCR_008520	FACS data analysis

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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
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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
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Source data 3 Raw data of western-blotting analysis.: https://cdn.elifesciences.org/articles/70151/elife-70151-data3-v3.zip
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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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Joji Nakayama
Lora Tan
Yan Li
Boon Cher Goh
Shu Wang
Hideki Makinoshima
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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A chemical screen for identification of epiboly-interrupting drugs.

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).

Epiboly could serve as a marker for this screening.

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

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

Pizotifen suppressed metastatic dissemination of MDA-MB-231 and MIA-PaCa2 cells in a zebrafish xenotransplantation model.

Pizotifen suppressed metastatic progression in a mouse model of metastasis.

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.

HTR2C induced epithelial-to-mesenchymal transition (EMT)-mediated metastatic dissemination of human cancer cells.

HTR2C promoted EMT-mediated metastatic dissemination of poorly metastatic human cancer cells in a zebrafish xenotransplantation model.

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

Quantification analyses of western blotting bands in Figure 5C.

Quantification analyses of western blotting bands in Figure 5D.

Quantification analyses of western blotting bands in Figure 5F.

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

Quantification analyses of western blotting bands in Figure 5H.

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

A list of the drugs that interfere with epiboly progression in zebrafish.

Primary targets of the identified drugs.

Effects of pharmacological inhibition of HTR2C on metastatic dissemination of MDA-MB-231 cells in zebrafish xenografted models.

Effects of pharmacological inhibition of HTR2C on metastatic dissemination of Mia-PaCa2 cells in zebrafish xenografted models.

Effects of genetic inhibition of HTR2C on metastatic dissemination of MDA-MB-231 cells in zebrafish xenografted models.

Effects of HTR2C overexpression on metastatic dissemination of MCF7 cells in zebrafish xenografted models.

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Source data 1

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