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

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.


Introduction 48
Metastasis, a leading contributor to the morbidity of cancer patients, occurs through 49 multiple steps: invasion, intravasation, extravasation, colonization, and metastatic tumor 50 formation (1-3). The physical translocation of cancer cells is an initial step of metastasis 51 and molecular mechanisms of it involve cell motility, the breakdown of local basement 52 membrane, loss of cell polarity, acquisition of stem cell-like properties, and EMT (4-6). 53 These cell-biological phenomena are also observed during vertebrate gastrulation in that 54 evolutionarily conserved morphogenetic movements of epiboly, internalization, 55 convergence, and extension progress (7). In zebrafish, the first morphogenetic movement, 56 epiboly, is initiated at approximately 4 hours post fertilization (hpf) to move cells from 57 the animal pole to eventually engulf the entire yolk cell by 10 hpf (8,9). The embryonic 58 cell movements are governed by the molecular mechanisms that are partially shared in 59 metastatic cell dissemination. 60 At least fifty common genes were shown to be involved in both metastasis and 61 gastrulation progression: Knockdown of these genes in Xenopus or zebrafish induced 62 gastrulation defects; conversely, overexpression of these genes conferred metastatic 63 potential on cancer cells and knockdown of these genes suppressed metastasis (Table S1). 64 This evidence led us to hypothesize that small molecules that interrupt zebrafish 65 gastrulation may suppress metastatic progression of human cancer cells. 66 Here we report a unique screening concept based on the hypothesis. Pizotifen, an 67 antagonist for HTR2C, was identified from the screen as a "hit" that interrupted zebrafish 68 gastrulation. A mouse model of metastasis confirmed pharmacological and genetic 69 inhibition of HTR2C suppressed metastatic progression. Moreover, HTR2C induced EMT 70 and promoted metastatic dissemination of non-metastatic cancer cells in a zebrafish 71 xenotransplantation model. These results demonstrated that this concept could offer a 72 novel high-throughput platform for discovery of anti-metastasis drugs and can be 73 converted to a chemical genetic screening platform. 74 nodules per lung in all 10 mice analyzed; conversely, Pizotifen-treated mice (n=10) 210 formed 0 to 5 nodules per lung in all 10 mice analyzed ( Figure 3D). Histological analyses 211 confirmed that metastatic lesions in the lungs were detected in all vehicle-treated mice; 212 conversely, they were detected in only 2 of 10 Pizotifen-treated mice and the rest of the 213 mice showed metastatic colony formations around the bronchiole of the lung. In addition, 214 4 of 10 vehicle-treated mice exhibited metastasis in the liver and the rest showed 215 metastatic colony formation around the portal tract of the liver. In contrast, none of 10 216 Pizotifen-treated mice showed liver metastases and only half of the 10 mice showed 217 metastatic colony formation around the portal tract ( Figure 3E). These results indicate that 218 Pizotifen can suppress metastasis progression without affecting primary tumor growth. 219 To eliminate the possibility that the metastasis suppressing effects of Pizotifen 220 might result from off-target effects, we conducted validation experiments to determine 221 whether knockdown of HTR2C would show the same effects. The basic experimental 222 process followed the experimental design described above except that sub-clones of 4T1 223 cells that expressed shRNA targeting either LacZ or HTR2C were injected into the MFP 224 of female BALB/c mice and the mice were maintained without drug. Histological 225 analyses revealed that all of the mice (n=5) that were inoculated with 4T1 cells expressing 226 shRNA targeting LacZ showed metastases in the lungs. The mean number of metastatic 227 lesions in a lung was 26.4±7.8. In contrast, only one of the mice (n=5) were inoculated 228 with 4T1 cells expressing shRNA targeting HTR2C showed metastases in the lungs and 229 the rest of the mice showed metastatic colony formation around the bronchiole of the 230 lung. The mean number of metastatic lesions in the lung significantly decreased to 10% of 231 those of mice that were inoculated with 4T1 cells expressing shRNA targeting LacZ 232 ( Figure 3F-H). 233 Taken together, pharmacological and genetic inhibition of HTR2C showed an 234 anti-metastatic effect in the 4T1 model system. 235 236 HTR2C promoted EMT-mediated metastatic dissemination of human cancer cells 237 Although pharmacological and genetic inhibition of HTR2C inhibited metastasis 238 progression, a role for HTR2C on metastatic progression has not been reported. 239 Therefore, we examined whether HTR2C could confer metastatic properties on poorly 240 metastatic cells. 241 Firstly, we established a stable sub-clone of MCF7 human breast cancer cells 242 expressing either vector control or HTR2C. Vector control expressing MCF7 cells 243 maintained highly organized cell-cell adhesion and cell polarity; however, HTR2C-244 expressing MCF7 cells led to loss of cell-cell contact and cell scattering. The cobblestone-245 like appearance of these cells was replaced by a spindle-like, fibroblastic morphology. 246 Western blotting and immunofluorescence (IF) analyses revealed that HTR2C-expressing 247 MCF7 cells showed loss of E-cadherin and EpCAM, and elevated expressions of N-248 cadherin, vimentin and an EMT-inducible transcriptional factor Zeb1. Similar effects 249 were validated through another experiment using an immortal keratinocyte cell line, 250 HaCaT cells, in that HTR2C-expressing HaCaT cells also showed loss of cell-cell contact 251 and cell scattering with loss of epithelial markers and gain of mesenchymal markers 252 ( Figure 4A-C). Therefore, both the morphological and molecular changes in the HTR2C-253 expressing MCF7 and HaCaT cells demonstrated that these cells had undergone an EMT. 254 Next, we examined whether HTR2C-driven EMT could promote metastatic 255 dissemination of human cancer cells. Boyden chamber assay revealed that HTR2C 256 expressing MCF7 cells showed an increased cell motility and invasion compared with 257 vector control-expressing MCF7 cells in vitro ( Figure 4D) compared with tumors from vehicle-treated mice ( Figure 5A-C). However, mesenchymal 289 markers did not change between vehicle and Pizotifen-treated MDA-MB-231 cells (data 290 not shown). We further analyzed E-cadherin positive (E-cad + ) cells in Pizotifen-treated 291 MDA-MB-231 cells. The E-cad + cells showed elevated expressions of epithelial markers 292 KRT14 and KRT19; and decreased expression of mesenchymal makers vimentin, MMP1, 293 MMP3, and S100A4. Recent research reports that an EMT program needs to be transient 294 and reversible and that a mesenchymal phenotype in cancer cells is achieved by 295 constitutive ectopic expression of Zeb1. In accordance with the research, the E-cad + cells 296 and 4T1 primary tumors from Pizotifen-treated mice had decreased Zeb1 expression 297 compared with vehicle-treated cells and tumors from vehicle-treated mice ( Figure 5D). In 298 contrast, HTR2C-expressing MCF7 and HuMEC cells expressed Zeb1 but not vehicle 299 control MCF7 and HuMEC cells ( Figure 4C). These results indicate that HTR2C-300 mediated signaling induced EMT through up-regulation of Zeb1 and blocking HTR2C 301 with Pizotifen induced mesenchymal to epithelial transition through downregulation of 302

Zeb1. 303
We further investigated the mechanism of action of how blocking HTR2C with 304 Pizotifen induced down-regulation of Zeb1. In embryogenesis, serotonin-mediated 305 signaling is required for Wnt-dependent specification of the superficial mesoderm during 306 gastrulation (23). In cancer cells, overexpression of HTR1D is associated with Wnt-307 signaling which enables induction of EMT (24,25). This evidence led to a hypothesis that 308 HTR2C-mediated signaling might turn on transcriptional activity of b-catenin and that 309 might induce up-regulation of EMT-TFs. IF analyses revealed b-catenin was accumulated 310 in the nucleus of HTR2C-expressing MCF7 cells but it was located in the cytoplasm of 311 vector control-expressing cells ( Figure 5E). Nuclear accumulation of b-catenin in 312 HTR2C-expressing MCF7 cells was confirmed by western blot ( Figure 5F). In contrast,    Table S6. 408 Reducing or eliminating mortality associated with metastatic disease is a key goal of 432 medical oncology, but few models exist that allow for rapid, effective screening of novel 433 compounds that target the metastatic dissemination of cancer cells. Based on accumulated 434 evidence that at least fifty genes play an essential role in governing both metastasis and 435 gastrulation progression (Table 1S), we hypothesized that small molecule inhibitors that 436 interrupt gastrulation of zebrafish embryos might suppress metastatic progression of 437 human cancer cells. We created a unique screening concept utilizing gastrulation of 438 zebrafish embryos to test the hypothesis. Our results clearly confirmed our hypothesis: 439 25.6% (20/76 drugs) of epiboly-interrupting drugs could also suppress cell motility and 440 invasion of highly metastatic human cell lines in vitro. In particular, Pizotifen which is an 441 antagonist for serotonin receptor 2C and one of the epiboly-interrupting drugs, could 442 suppress metastasis in a mouse model ( Figure 3A-E). Thus, this screen could offer a novel 443 platform for discovery of anti-metastasis drugs. 444 There are at least two advantages to the screen described herein. One is that the 445 screen can easily be converted to a chemical genetic screening platform. Indeed, we have 446 provided the first evidence that HTR2C, which is a primary target of Pizotifen, induced 447 EMT and promoted metastatic dissemination of cancer cells ( Figure 4A-E). In this 448 research, 1,280 FDA approval drugs were screened, this is less than a few percent of all of 449 druggable targets (approximately 100 targets) in the human proteome in the body. If 450 chemical genetic screening using specific inhibitor libraries were conducted, more genes 451 that contribute to metastasis and gastrulation could be identified. The second advantage is 452 that the screen enables one researcher to test 100 drugs in 5 hours with using optical 453 microscopy, drugs, and zebrafish embryos. That indicates this screen is not only highly 454 efficient, low-cost, and low-labor but also enables researchers who do not have high 455 throughput screening instruments to conduct drug screening for anti-metastasis drugs. University, Boston, MA). All culture methods followed the supplier's instruction. Cell 496 viability assay was performed as previously described (16) Western blotting was performed as described previously (16). Anti-PRMT1, anti-505 CYP11A1, anti-E-cadherin, anti-EpCAM, anti-Vimentin, anti-N-cadherin, anti-Zeb1, 506 anti-Histone H3, anti-a-tubulin and anti-GAPDH antibodies were purchased from Cell 507 signaling Technology. Anti-HTR2C and anti-DRD2 antibodies were purchased form 508 Abcam. Anti-phospho-GSK3b (Ser9), anti-GSK3b, Anti-KRT18, anti-KRT19, anti-509 MMP1, anti-MMP2, anti-S100A4, anti-Luciferase, and anti-b-catenin antibodies were 510 purchased from Santa Cruz. 511 512 Flow cytometry 513 Cells were stained with FITC-conjugated E-cadherin antibody (Biolegend, San Diego, 514 CA). Flow cytometry was performed as described (26)  Immunofluorescence microscopy assay was performed by previously described (16). Goat 530 anti-mouse and goat anti-rabbit immunoglobulin G (IgG) antibodies conjugated to Alexa 531 Fluor 488 (Life Technologies) and diluted at 1:100 were used. Nuclei were visualized by 532 the addition of 2µg/ml of 4', 6-diamidino-2-phenylindole (DAPI) and photographed at 533 100x magnification by a fluorescent microscope BZ-X700 (KEYENCE, Japan). 534 535 Boyden chamber cell motility and invasion assay 536    704 Table S1. 705 A list of the fifty genes that play essential role in governing both metastasis and 706 gastrulation progression. The gastrulation defects in Xenopus or zebrafish that are 707 induced by knockdown of each of these genes, were indicated. The molecular 708 mechanism in metastasis that are inhibited by knockdown of each of the same genes, 709 were indicated. 710  Table S2. A list of the drugs that interfere with epiboly progression in zebrafish. 714 Related to Figure 1.

724
Related to Figure 2D.

733
Related to Figure S3. 734 The numbers and frequencies of the fish showing the dissemination patterns in vehicle 735 or Pizotifen-treated group, were indicated. The fish showed both patterns of 736 dissemination were redundantly counted in this analysis. 737

741
Related to Figure 2E.