A zebrafish embryo screen utilizing gastrulation identifies the HTR2C inhibitor pizotifen as a suppressor of EMT-mediated metastasis
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Evaluation Summary:
This manuscript develops a novel approach using the zebrafish to identify suitable drugs against the spread of cancer. With some stronger support of the methodology and conclusions, it will be of interest to cancer biologists, developmental biologists, and pharmacologists.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Abstract
Metastasis is responsible for approximately 90% of cancer-associated mortality but few models exist that allow for rapid and effective screening of anti-metastasis drugs. Current mouse models of metastasis are too expensive and time consuming to use for rapid and high-throughput screening. Therefore, we created a unique screening concept utilizing conserved mechanisms between zebrafish gastrulation and cancer metastasis for identification of potential anti-metastatic drugs. We hypothesized that small chemicals that interrupt zebrafish gastrulation might also suppress metastatic progression of cancer cells and developed a phenotype-based chemical screen to test the hypothesis. The screen used epiboly, the first morphogenetic movement in gastrulation, as a marker and enabled 100 chemicals to be tested in 5 hr. The screen tested 1280 FDA-approved drugs and identified pizotifen, an antagonist for serotonin receptor 2C (HTR2C) as an epiboly-interrupting drug. Pharmacological and genetic inhibition of HTR2C suppressed metastatic progression in a mouse model. Blocking HTR2C with pizotifen restored epithelial properties to metastatic cells through inhibition of Wnt signaling. In contrast, HTR2C induced epithelial-to-mesenchymal transition through activation of Wnt signaling and promoted metastatic dissemination of human cancer cells in a zebrafish xenotransplantation model. Taken together, our concept offers a novel platform for discovery of anti-metastasis drugs.
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Author Response:
Reviewer #1 Public Review:
Nakayama and colleagues report a unique screening concept utilizing conserved mechanisms between zebrafish gastrulation and cancer metastasis for identification of potential anti-metastatic drugs. They screen 1280 FDA-approved drugs using the gastrulation as a marker, and identify Pizotifen as an epiboly interrupting drug. Then they find that pharmacologic and genetic inhibition of HTR2C, a target of Pizotifen, suppresses metastatic progression in a zebrafish and mouse model through inhibition of epithelial to mesenchymal transition (EMT) via Wnt-signaling.
Their work is of interest and has the potential to appeal to a broad audience. However, additional experiments are needed to further substantiate their concept that human cancer metastasis mimic/recapitulate zebrafish gastrulation in …
Author Response:
Reviewer #1 Public Review:
Nakayama and colleagues report a unique screening concept utilizing conserved mechanisms between zebrafish gastrulation and cancer metastasis for identification of potential anti-metastatic drugs. They screen 1280 FDA-approved drugs using the gastrulation as a marker, and identify Pizotifen as an epiboly interrupting drug. Then they find that pharmacologic and genetic inhibition of HTR2C, a target of Pizotifen, suppresses metastatic progression in a zebrafish and mouse model through inhibition of epithelial to mesenchymal transition (EMT) via Wnt-signaling.
Their work is of interest and has the potential to appeal to a broad audience. However, additional experiments are needed to further substantiate their concept that human cancer metastasis mimic/recapitulate zebrafish gastrulation in terms of conserved mechanism, as well as to confirm the validity of their screening method regarding to the effects of global toxicity.
Major concerns:
The first major concern I have is the appropriateness to think the gastrulation as a parameter/index of cancer metastasis. While they cherry-picked some genes that they are known to be involved in both gastrulation and cancer metastasis, more broad analysis should probably be necessary to conclude so. For examples, the authors can analyze comprehensive RNA-seq data set to see if the pathways/networks are similar between gastrulation (zebrafish embryo development data set) and cancer metastasis (benign/primary tumors vs metastasis tumors in TCGA).
The conservation of embryonic EMT and tumor metastasis EMT has long been well recognized. Now we cited some of these published references (Nieto et al., 2016; Thiery et al., 2009; Yang and Weinberg, 2008). In Table 1, we compiled 50 genes based on published literature to provide further and strong evidence to support this conservation. Knockdown of these genes in Xenopus or zebrafish induced gastrulation defects; conversely, overexpression of these genes conferred metastatic potential on cancer cells and knockdown of these genes suppressed metastasis. Although this point is not really an objective of this study, we believe that the evidence for the conservation is sufficiently convincing to provide the basis for our study. Further RNA-seq comparison of zebrafish embryonic EMT and human tumor metastasis should be beyond the scope of the current study. Generally, the transcriptomic data for zebrafish embryo development at the epiboly/gastrulation stage are based on the whole embryos which include all other activities and are not specific to EMT; thus, it may not be a proper comparison with tumor metastasis data to search for more evidence.
The second concern is about the Pizotifen's effects on cancer metastasis. Since the Pizotifen suppresses gastrulation, it might have some harmful effect on the organogenesis/development of day2 embryos that they used in zebrafish transplantation model. And if so, cancer metastasis can be suppressed indirectly. The authors could examine if Pizotifen could have some side effects on day2 embryos. The drug also has some cell viability suppressive effects in vivo so as the pics in Fig.2D looks like, and it would be good if this possibility was excluded.
We had not observed any abnormality in development of Tg (kdrl;GFP) and WT zebrafish at day 2 when these fish were treated with 5µM Pizotifen. However, more than 20µM Pizotifen treatment affected approximately 10% of these fish. The affected fish show shorter tail rather than that of vehicle-treated zebrafish. In xenograft experiments, zebrafish embryos at day 2 were treated with 5µM Pizotifen. The concentration of Pizotifen did not affected development of day2 embryos.
Futhermore, we demonstrated Pizotifen did not affect primary tumor growth in a mice model of metastasis using 4T1 cells by two different experimental methods. One is that tumor measurement revealed that the sizes of the primary tumors in Pizotifen-treated mice were equal to those in the vehicle-treated mice at the time of resection on day 10 post inoculation. The other is that IF-staining showed the percentage of Ki67 positive cells in the resected primary tumors of Pizotifen-treated mice were the same as those of vehicle-treated mice (Figure 3A and B). Therefore, we conclude that Pizotifen suppress metastasis without affecting cell viability in vivo.
Finally, the mechanistic parts would need more confirmation and rescue experiments. Transplanted cells can be sorted after the treatment and the expression changes of EMT markers can be examined to see if the phenomenon happens in vivo as well. All main results can be rescued to see if the effect of Pizotifen against EMT happens through HTR2C-Wnt axis.
Figure 5C showed that 4T1 primary tumors from Pizotifen-treated mice has elevated E95 cadherin expression compared with tumors from vehicle-treated mice. Furthermore, Figure 5C also demonstrated that β-catenin accumulated in the nucleus, and phospho-GSKβ and Zeb1 expression were decreased in 4T1 primary tumors from Pizotifen-treated mice compared with vehicle-treated mice. Loss of E-cadherin plays an essential role in promoting EMT-mediated metastasis since loss of E-cadherin itself is enough to promote metastasis. In contrast, overexpression of mesenchymal markers: vimentin and N-cadherin is not sufficient to induce metastasis. Based on our data from Figure 5C and the accumulated evidences, we conclude that Pizotifen restored epithelial properties to metastatic cells through a decrease of transcriptional activity of β-catenin in vivo
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Evaluation Summary:
This manuscript develops a novel approach using the zebrafish to identify suitable drugs against the spread of cancer. With some stronger support of the methodology and conclusions, it will be of interest to cancer biologists, developmental biologists, and pharmacologists.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Reviewer #1 (Public Review):
Nakayama and colleagues report a unique screening concept utilizing conserved mechanisms between zebrafish gastrulation and cancer metastasis for identification of potential anti-metastatic drugs. They screen 1280 FDA-approved drugs using the gastrulation as a marker, and identify Pizotifen as an epiboly interrupting drug. Then they find that pharmacologic and genetic inhibition of HTR2C, a target of Pizotifen, suppresses metastatic progression in a zebrafish and mouse model through inhibition of epithelial to mesenchymal transition (EMT) via Wnt-signaling.
Their work is of interest and has the potential to appeal to a broad audience. However, additional experiments are needed to further substantiate their concept that human cancer metastasis mimic/recapitulate zebrafish gastrulation in terms of conserved …
Reviewer #1 (Public Review):
Nakayama and colleagues report a unique screening concept utilizing conserved mechanisms between zebrafish gastrulation and cancer metastasis for identification of potential anti-metastatic drugs. They screen 1280 FDA-approved drugs using the gastrulation as a marker, and identify Pizotifen as an epiboly interrupting drug. Then they find that pharmacologic and genetic inhibition of HTR2C, a target of Pizotifen, suppresses metastatic progression in a zebrafish and mouse model through inhibition of epithelial to mesenchymal transition (EMT) via Wnt-signaling.
Their work is of interest and has the potential to appeal to a broad audience. However, additional experiments are needed to further substantiate their concept that human cancer metastasis mimic/recapitulate zebrafish gastrulation in terms of conserved mechanism, as well as to confirm the validity of their screening method regarding to the effects of global toxicity.
Major concerns:
The first major concern I have is the appropriateness to think the gastrulation as a parameter/index of cancer metastasis. While they cherry-picked some genes that they are known to be involved in both gastrulation and cancer metastasis, more broad analysis should probably be necessary to conclude so. For examples, the authors can analyze comprehensive RNA-seq data set to see if the pathways/networks are similar between gastrulation (zebrafish embryo development data set) and cancer metastasis (benign/primary tumors vs metastasis tumors in TCGA).
The second concern is about the Pizotifen's effects on cancer metastasis. Since the Pizotifen suppresses gastrulation, it might have some harmful effect on the organogenesis/development of day2 embryos that they used in zebrafish transplantation model. And if so, cancer metastasis can be suppressed indirectly. The authors could examine if Pizotifen could have some side effects on day2 embryos. The drug also has some cell viability suppressive effects in vivo so as the pics in Fig.2D looks like, and it would be good if this possibility was excluded.
Finally, the mechanistic parts would need more confirmation and rescue experiments. Transplanted cells can be sorted after the treatment and the expression changes of EMT markers can be examined to see if the phenomenon happens in vivo as well. All main results can be rescued to see if the effect of Pizotifen against EMT happens through HTR2C-Wnt axis.
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Reviewer #2 (Public Review):
The manuscript by Nakayama describes the use of a small molecule based screen in zebrafish embryos to find compounds that selectively impact metastasis in cancer. This screen is based on the strong premise that compounds that selectively delay or prevent gastrulation where cells are extensively migrating and undergoing convergence and extension movements in zebrafish embryos are similar to the mechanisms co-opted by malignant cells during metastasis. Screening 1280 FDA approved drug from the Prestwick library identified a total of 132 hits which delayed or disrupted epiboly. After eliminating toxic compounds and drugs that severely disrupted gastrulation, 5.2% of the drugs delayed epiboly. 20 of the compounds identified decreased cancer cell migration, and one compound, Pitzotifen, was shown to inhibit cell …
Reviewer #2 (Public Review):
The manuscript by Nakayama describes the use of a small molecule based screen in zebrafish embryos to find compounds that selectively impact metastasis in cancer. This screen is based on the strong premise that compounds that selectively delay or prevent gastrulation where cells are extensively migrating and undergoing convergence and extension movements in zebrafish embryos are similar to the mechanisms co-opted by malignant cells during metastasis. Screening 1280 FDA approved drug from the Prestwick library identified a total of 132 hits which delayed or disrupted epiboly. After eliminating toxic compounds and drugs that severely disrupted gastrulation, 5.2% of the drugs delayed epiboly. 20 of the compounds identified decreased cancer cell migration, and one compound, Pitzotifen, was shown to inhibit cell invasion and metastasis in cell based and in vivo xenograft based assays in both zebrafish and mice. Pitzotifen is an antagonist for the serotonin receptor 2C (HTR2C) which the authors show is a driver of ZEB1 mediated epithelial to mesenchymal transition (EMT) through regulation of Wnt/ beta-catenin signaling.
Strengths
1. The in vivo screening method in zebrafish is able to identify compounds that selectively effect gastrulation and consequently in cell lines have a pronounced effects on cell migration and invasion and Pizotifen also disrupts metastasis in both a zebrafish and murine xenograft assays. The assays especially in the zebrafish model arerong and convincing. This study further supports current findings that show that cancer cells co-opt normal developmental programs to spread and metastasize.
2. A clear logical progression of experiments is provided to demonstrate that Pizotifen through regulation of Wnt-signaling can ablate EMT mediated by ZEB1.
3. Pizotifen is an inhibitor of serotonin receptor HTR2C which can regulate EMT, metastases and ZEB1 expression.
Weaknesses
1. The significance of the genes/ proteins identified in the screen are not well articulated. Did the screen identify genes that are correlated with poor outcome and or metastasis in cancer. Second, the selection of human cancer cell lines to validate the findings are not clear. Is HTR2C and other genes identified relevant only to the subset of cancers that the cell lines represent or is it a common pan-cancer effect. Analyses of TCGA or other data sets to show relevance of HTR2C gene expression and survival and or metastasis would greatly strengthen the relevance of the hits and the rationale for picking the cell lines to study.
2. Some of the data provided does not support the conclusions drawn. For example, in figure 3 C, the luciferase images clearly show that Pizotifen treated mice have smaller tumors while the data in 3 A and B do not show this data. The measurements of tumor volume appear to be taken too early to actually be reproducible/representative.
3. It is not clear if HTR2C selectively only inhibits ZEB1 mediated metastases or are its effects more general i.e. its expression also effects other EMT regulators for example SNAIL, SLUG and or TWIST expression.
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Reviewer #3 (Public Review):
Nakayama et al. applied the molecular similarities between metastatic cell movement and cell movement in gastrulation to conduct a screening using FDA-approved drugs. Using a zebrafish model involving epiboly gastrulation as a marker, in vitro human cancer cell lines, xenotransplantation, and mouse models, they find that Pizotifen, an antagonist for serotonin receptor 2C (HTR2C), has anti-metastatic effects. The authors further show that HTR2C overexpression is sufficient to induce metastasis. They further link this finding to the wnt pathway, suggesting that the decrease of ß-catenin is involved in the loss of metastatic properties. Overall, the data is interesting and strong. However, the link to the wnt pathway is weak without sufficient experimental evidence.
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