Tumor Metastasis: Fishing for drugs

About 90% of all cancer-related deaths are caused by metastasis, which is when cancer cells spread to other parts of the body to form new tumors. Yet, the majority of currently available therapeutics do not inhibit metastasis, and only target the primary tumor where the cancer initially arises from.

To screen anti-cancer drugs, researchers often carry out experiments on mice or cells grown in the laboratory. While these model systems have led to effective treatments, they have limitations when it comes to testing drugs that block metastasis. For instance, cells cultured in the laboratory cannot accurately replicate tumor progression in humans, and metastasis can take at least several weeks to appear in mouse models, which are expensive to create and maintain. Now, in eLife, Joji Nakayama, Zhiyuan Gong and co-workers report an innovative zebrafish model for screening anti-metastasis drugs.

The zebrafish was introduced to the research field in 1972, and has become a powerful model system for cancer research, due to its relative transparency, high reproduction rates, and genetic similarity to humans. Early in development, cells in the zebrafish embryo undergo a morphological change and migrate inwards via a process called epiboly. The way these healthy cells move is similar to how cancer cells travel across tissues during metastasis. Hence, Nakayama et al. proposed that small-molecule inhibitors that interrupt epiboly may also suppress metastasis.

The screening platform created by Nakayama et al. makes it easy to rapidly find new drugs that suppress metastasis, while circumventing the limitations of cell culture and mouse model systems. In addition, zebrafish injected with human cancer cells can serve as an additional means for narrowing down which drugs to test in mouse models. Having zebrafish join the drug discovery platform will hopefully result in more and better treatments for patients with metastatic cancers.

https://doi.org/10.7554/eLife.76632

Gastrulation Screening to Identify Anti-metastasis Drugs in Zebrafish Embryos    

Few models exist that allow for rapid and effective screening of anti-metastasis drugs. Here, we present a drug screening protocol utilizing gastrulation of zebrafish embryos for identification of anti-metastasis drugs. Based on the evidence that metastasis proceeds through utilizing the molecular mechanisms of gastrulation, we hypothesized that chemicals interrupting zebrafish gastrulation might suppress the metastasis of cancer cells. Thus, we developed a phenotype-based chemical screen that uses epiboly, the first morphogenetic movement in gastrulation, as a marker. The screen only needs zebrafish embryos and enables hundreds of chemicals to be tested in five hours by observing the epiboly progression of chemical-treated embryos. In the screen, embryos at the two-cell stage are firstly corrected and then developed to the sphere stage. The embryos are treated with a test chemical and incubated in the presence of the chemical until vehicle-treated embryos develop to the 90% epiboly stage. Finally, positive ‘hit’ chemicals that interrupt epiboly progression are selected by comparing epiboly progression of the chemical-treated and vehicle-treated embryos under a stereoscopic microscope. A previous study subjected 1,280 FDA-approved drugs to the screen and identified Adrenosterone and Pizotifen as epiboly-interrupting drugs. These were validated to suppress metastasis of breast cancer cells in mice models of metastasis. Furthermore, 11β-hydroxysteroid dehydrogenase 1 (HSD11β1) and serotonin receptor 2C (HTR2C), the primary targets of adrenosterone and pizotifen, respectively, promoted metastasis through induction of epithelial-mesenchymal transition (EMT). This screen can also measure suppressor effect of crude drugs. We subjected 120 herbal medicines to this screen and identified cinnamon bark extract as an epiboly-interfering drug. Cinnamon bark extract was validated to suppress metastatic dissemination of breast cancer cells in a zebrafish xenograft model. Therefore, this screen could be converted into a chemical genetic screening platform for identification of metastasis-promoting genes.

https://doi.org/10.21769/BioProtoc.4525

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.

https://doi.org/10.7554/eLife.70151

A Novel Zebrafish Model of Metastasis Identifies the HSD11β1 Inhibitor Adrenosterone as a Suppressor of Epithelial–Mesenchymal Transition and Metastatic Dissemination

Metastasis of cancer cells is multi-step process and dissemination is an initial step. Here we report a tamoxifen-controllable Twist1a-ER^T2 transgenic zebrafish line as a new animal model for metastasis research, and demonstrate that this model can serve as a novel platform for discovery of antimetastasis drugs targeting metastatic dissemination of cancer cells. By crossing Twist1a-ER^T2 with xmrk (a homolog of hyperactive form of EGFR) transgenic zebrafish, which develops hepatocellular carcinoma, approximately 80% of the double transgenic zebrafish showed spontaneous cell dissemination of mCherry-labeled hepatocytes from the liver to the entire abdomen region and the tail region. The dissemination is accomplished in 5 days through induction of an epithelial-to-mesenchymal transition. Using this model, we conducted in vivo drug screening and identified three hit drugs. One of them, adrenosterone, an inhibitor for hydroxysteroid (11-beta) dehydrogenase 1 (HSD11β1), has a suppressor effect on cell dissemination in this model. Pharmacologic and genetic inhibition of HSD11β1 suppressed metastatic dissemination of highly metastatic human cell lines in a zebrafish xenotransplantation model. Through downregulation of Snail and Slug, adrenosterone-treated cells recovered expression of E-cadherin and other epithelial markers and lost partial expression of mesenchymal markers compared with vehicle-treated cells. Taken together, our model offers a useful platform for the discovery of antimetastasis drugs targeting metastatic dissemination of cancer cells.

https://doi.org/10.1158/1541-7786.MCR-19-0759

Cinnamon bark extract suppresses metastatic dissemination of cancer cells through inhibition of glycolytic metabolism

Metastasis is responsible for approximately 90% of cancer-associated mortality and proceeds through multiple steps. Several herbal medicines are reported to inhibit primary tumor growth, but the suppressor effects of the medicines on metastasis progression are still not fully elucidated. Here we report that cinnamon bark extract (CBE) has a suppressor effect on metastatic dissemination of cancer cells. Through a phenotypic screening using zebrafish embryos, CBE was identified to interfere with the gastrulation progression of zebrafish embryos, of which the molecular mechanisms are conserved in metastasis progression. A Boyden chamber assay showed that CBE decreased cell motility and invasion of MDA-MB-231 human breast cancer cells without affecting their cell viability. Furthermore, CBE suppressed metastatic dissemination of the cells in a zebrafish xenotransplantation model. Quantitative metabolome analyses revealed that the productions of glucose-6-phosphate (G6P) and fructose 6-phosphate which are intermediate metabolites of glycolytic metabolism were interrupted in CBE-treated cells. qPCR and western-blotting analyses revealed that CBE-treated cells showed decreased expression of hexokinase 2 (HK2) which yields G6P. Pharmacological inhibition of HK2 with 2-deoxy-D-glucose suppressed cell invasion and migration of the cells without affecting their cell viability. Taken together, CBE suppresses metastatic dissemination of cancer cells through inhibition of glycolysis metabolism.

https://doi.org/10.1007/s11418-022-01624-3

Interestingly, both HTR2C and HSD11β1 are associated with stress hormones.