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.

The microtubule-associated protein HURP recruits the centrosomal protein TACC3 to regulate K-fiber formation and support chromosome congression.

Kinetochore fibers (K-fibers) are microtubule bundles attached to chromosomes. Efficient K-fiber formation is required for chromosome congression, crucial for faithful chromosome segregation in cells. However, the mechanisms underlying K-fiber formation before chromosome biorientation remain unclear. Depletion of hepatoma up-regulated protein (HURP), a RanGTP-dependent microtubule-associated protein localized on K-fibers, has been shown to result in low-efficiency K-fiber formation. Therefore, here we sought to identify critical interaction partners of HURP that may modulate this function. Using co-immunoprecipitation and bimolecular fluorescence complementation assays, we determined that HURP interacts directly with the centrosomal protein transforming acidic coiled coil-containing protein 3 (TACC3), a centrosomal protein, both in vivo and in vitro through the HURP1-625 region. We found that HURP is important for TACC3 function during kinetochore microtubule assembly at the chromosome region in prometaphase. Moreover, HURP regulates stable lateral kinetochore attachment and chromosome congression in early mitosis by modulation of TACC3. These findings provide new insight into the coordinated regulation of K-fiber formation and chromosome congression in prometaphase by microtubule-associated proteins.

HURP regulates chromosome congression by modulating kinesin Kif18A function.

Chromosome biorientation and congression during mitosis require precise control of microtubule dynamics [1-4]. The dynamics of kinetochore microtubules (K-MTs) are regulated by a variety of microtubule-associated proteins (MAPs) [4-9]. Recently, a MAP known as HURP (hepatoma upregulated protein) was identified [10-12]. During mitosis, Ran-guanosine 5'-triphosphate (RanGTP) releases HURP from the importin ß inhibitory complex and allows it to localize to the kinetochore fiber (k-fiber) [12, 13]. HURP stabilizes k-fibers and promotes chromosome congression [12, 14, 15]. However, the molecular mechanism underlying the role of HURP in regulating chromosome congression remains elusive. Here, we show that overexpression of the N-terminal microtubule binding domain (1-278 aa, HURP(278)) of HURP induces a series of mitotic defects that mimic the effects of Kif18A depletion. In addition, coimmunoprecipitation and bimolecular fluorescence complementation assays identify Kif18A as a novel interaction partner of HURP. Furthermore, quantitative results from live-cell imaging analyses illustrate that HURP regulates Kif18A localization and dynamics at the plus end of K-MTs. Lastly, misaligned chromosomes in HURP(278)-overexpressing cells can be partially rescued by the overexpression of Kif18A. Our results demonstrate in part the regulatory mechanism for Kif18A during chromosome congression and provide new insights into the mechanism of chromosome movement at the metaphase plate.