Blanka Leber

Proteins Required for Centrosome Clustering in Cancer Cells

Identified in an RNA interference screen, proteins that prevent spindle multipolarity in human cancer cells may prove to be promising drug targets. Releasing Tension Could Help Tame Tumor Cells Tumor cells do some things very well—proliferate, for example—but do other things very badly. Instead of orderly, bipolar cytoskeletal machinery that neatly pulls the chromosomes apart during cell division, tumor cells are often multipolar, a state caused by extra copies of the microtubule organizer, the centrosome. These extra organizing centers form multipolar spindle apparatuses, the scaffolds on which cell division takes place, thus producing abnormal division and cell death. Some cells successfully correct this defect by bundling the extra spindles along with the primary ones, and these cells go on to divide properly and proliferate, becoming thriving tumors. Leber and colleagues have taken advantage of this organizational weakness of tumor cells and have identified the proteins these cells use to bundle their spindles next to the primary ones. These proteins are prime targets for new chemotherapeutic drugs, which would prevent corrective bundling, sentencing the cells to abnormal cell division and death. The authors used a small interfering RNA screen to eliminate the expression of 21,000 genes, one by one, in a squamous cell carcinoma cell line. By finding which cells had more than the usual two spindle poles during mitosis—indicative of extra centrosomes—and then verifying these in a second screen, the authors identified 82 genes that encode proteins that the cancer cells use to inhibit damaging multiple centrosomes. Among these proteins are many involved in attaching chromosomes to the cytoskeletal scaffold: those in the chromosomal passenger complex, a regulator of chromosome-microtubule interactions and spindle tension at the point where the spindle attaches; members of the Ndc80 complex, a contact point for chromosome-microtubule attachment; and CENPT, part of a DNA binding complex at the attachment point. Another particularly interesting protein picked up by the screen is CEP164, which is found at the ends of centriole appendages, which are microtubule anchors at the centrosome. By looking for commonalities among these proteins, the authors hypothesized that spindle tension might be a key characteristic required for effective corrective spindle bundling. They tested this idea by measuring the distance between the chromosomes, confirming a central role for tension in this homeostatic mechanism, by which cancer cells compensate for extra centrosomes. Therefore, interfering with the function of any of the proteins found in the screen could, in principle, eliminate this tension and prevent bundling, leading to tumor cell death. These potential drug targets that the cell uses to bypass normal protective mechanisms against cancer are new weapons that can be added to the arsenal of anticancer drug developers. Current cancer chemotherapies are limited by the lack of tumor-specific targets, which would allow for selective eradication of malignant cells without affecting healthy tissues. In contrast to normal cells, most tumor cells contain multiple centrosomes, which tend to cause the formation of multipolar mitotic spindles, chromosome segregation defects, and cell death. Nevertheless, many cancer cells divide successfully because they can cluster multiple centrosomes into two spindle poles. Inhibition of this centrosomal clustering, with consequent induction of multipolar spindles and subsequent cell death, would specifically target cancer cells and overcome one limitation of current cancer treatments. We have performed a genome-wide RNA interference screen to identify proteins involved in the prevention of spindle multipolarity in human cancer cells with supernumerary centrosomes. The chromosomal passenger complex, Ndc80 microtubule-kinetochore attachment complex, sister chromatid cohesion, and microtubule formation via the augmin complex were identified as necessary for centrosomal clustering. We show that spindle tension is required to cluster multiple centrosomes into a bipolar spindle array in tumor cells with extra centrosomes. These findings may explain the specificity of drugs that interfere with spindle tension for cancer cells and provide entry points for the development of therapeutics.

GF-15, a novel inhibitor of centrosomal clustering, suppresses tumor cell growth in vitro and in vivo

In contrast to normal cells, malignant cells are frequently aneuploid and contain multiple centrosomes. To allow for bipolar mitotic division, supernumerary centrosomes are clustered into two functional spindle poles in many cancer cells. Recently, we have shown that griseofulvin forces tumor cells with supernumerary centrosomes to undergo multipolar mitoses resulting in apoptotic cell death. Here, we describe the characterization of the novel small molecule GF-15, a derivative of griseofulvin, as a potent inhibitor of centrosomal clustering in malignant cells. At concentrations where GF-15 had no significant impact on tubulin polymerization, spindle tension was markedly reduced in mitotic cells upon exposure to GF-15. Moreover, isogenic cells with conditional centrosome amplification were more sensitive to GF-15 than parental controls. In a wide array of tumor cell lines, mean inhibitory concentrations (IC(50)) for proliferation and survival were in the range of 1 to 5 μmol/L and were associated with apoptotic cell death. Importantly, treatment of mouse xenograft models of human colon cancer and multiple myeloma resulted in tumor growth inhibition and significantly prolonged survival. These results show the in vitro and in vivo antitumor efficacy of a prototype small molecule inhibitor of centrosomal clustering and strongly support the further evaluation of this new class of molecules.

Abstract LB-294: Development and characterization of novel orally available hypoxia-inducible factor (HIF) signaling inhibitors as dual-mechanism cancer therapeutics

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC The HIF signaling pathway is a crucial way in which tumors can circumvent the constraints of regions of low oxygen (hypoxia) to induce angiogenesis and maintain proliferation. The oxygen regulated subunit of the transcription factor hypoxia-inducible factor 1 (HIF1), HIF1αlpha, is a positive factor in tumor growth and its expression has been correlated with poor patient prognosis in a number of settings. We here present in vitro and in vivo data for a novel series of orally available small-molecule HIF signaling modulators that show nanomolar inhibition of the HIF signaling pathway in addition to potent anti-proliferative activity against a large number of cell lines derived from solid and blood tumors (EC50 in the range 1-100nM). Phenotypically, the compounds elicit an initial G2/M arrest, followed by the induction of caspase-3/7 and the onset of apoptosis. The in vitro results also translate into in vivo animal models. The lead compounds from the series show efficacy in tumor xenograft mouse models, with dose-dependent tumor growth inhibition of 60-70% after oral dosing (MDA-MB-231 xenograft). Structural optimisation has additionally allowed us to improve the PK and physicochemical characteristics of the compounds, with a lead candidate currently in formal pre-clinical development with the aim of entering a Phase 1 clinical trial in multiple myeloma at the end of 2010. In conclusion, we believe that the development towards clinical Proof-of-Concept of this new class of dual-mechanism inhibitors of HIF signaling and cell proliferation presents a promising new option for cancer therapeutics. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-294.