The centrosomal protein TACC3 controls paclitaxel sensitivity by modulating a premature senescence program.

Microtubule-interfering cancer drugs such as paclitaxel (PTX) often cause chemoresistance and severe side effects, including neurotoxicity. To explore potentially novel antineoplastic molecular targets, we investigated the cellular response of breast carcinoma cells to short hairpin(sh)RNA-mediated depletion of the centrosomal protein transforming acidic coiled coil (TACC) 3, an Aurora A kinase target expressed during mitosis. Unlike PTX, knockdown of TACC3 did not trigger a cell death response, but instead resulted in a progressive loss of the pro-apoptotic Bcl-2 protein Bim that links microtubule integrity to spindle poison-induced cell death. Interestingly, TACC3-depleted cells arrested in G1 through a cellular senescence program characterized by the upregulation of nuclear p21(WAF), downregulation of the retinoblastoma protein and extracellular signal-regulated kinase 1/2, formation of HP1γ (phospho-Ser83)-positive senescence-associated heterochromatic foci and increased senescence-associated ß-galactosidase activity. Remarkably, the onset of senescence following TACC3 knockdown was strongly accelerated in the presence of non-toxic PTX concentrations. Thus, we conclude that mitotic spindle stress is a major trigger of premature senescence and propose that the combined targeting of the centrosomal Aurora A-TACC3 axis together with drugs interfering with microtubule dynamics may efficiently improve the chemosensitivity of cancer cells.

TACC3 depletion sensitizes to paclitaxel-induced cell death and overrides p21WAF-mediated cell cycle arrest.

Regulators of the mitotic spindle apparatus are attractive cellular targets for antitumor therapy. The centrosomal protein transforming acidic coiled coil (TACC) 3 is required for spindle assembly and proper chromosome segregation. In this study, we employed an inducible RNA interference approach to downregulate TACC3 expression. We show that TACC3 knock-down in NIH3T3 fibroblasts caused aneuploidy, but failed to overtly impair mitotic progression. TACC3 depletion rather triggered a postmitotic p53-p21(WAF) pathway and led to a reversible cell cycle arrest. Similar effects were induced by low concentrations of paclitaxel, a spindle poison used in antitumor therapy. Interestingly, however, and unlike in TACC3-proficient cells, paclitaxel was able to induce strong polyploidy and subsequent apoptosis in TACC3-depleted cells. Even though paclitaxel treatment was associated with the activation of the survival kinase Akt and an antiapoptotic expression of cytoplasmic p21(WAF) and cyclin D1, this inhibition of cell death was abrogated by depletion of TACC3. Thus, our data identify TACC3 as a potential target to overcome p21(WAF)-associated protection of transformed cells against paclitaxel-induced cell death.

Modulation of the activation status of Stat5a during LIF-induced differentiation of M1 myeloid leukemia cells.

Treatment of M1 myeloid leukemia cells with leukemia inhibitory factor (LIF) causes activation of transcription factors Stat1, Stat3 and Stat5a (signal transducers and activators of transcription). DNA-binding of Stat proteins was detectable for extended periods of time in LIF-treated M1 cells, which simultaneously underwent terminal differentiation. The relative composition of Stat factors in the protein-DNA complexes changed during time. Whereas Stat3 was activated up to 36 h during treatment with LIF, Stat5a was activated only short-termed. Similarly, high expression of the immediate early gene CIS (cytokine-inducible SH2-containing protein), a known target gene of Stat5 in hematopoietic cells, occurred only during the onset of differentiation. This suggests a role of Stat5a in the early phase of LIF-induced differentiation and growth arrest of M1 cells.

Induction of differentiation by IL-6-type cytokines is impaired in myeloid leukaemia cells unable to activate Stat5a.

The block of differentiation in myeloid leukaemia can be overcome by treatment with a variety of agents including cytokines. Interleukin 6 (IL-6) and leukaemia inhibitory factor (LIF) induce macrophage differentiation and growth arrest through activation of the Janus kinase (Jak)/signal transducers and activators of transcription (Stat) signal pathway in murine M1 myeloid leukaemia cells. Treatment of various other myeloid leukaemia lines with LIF or IL-6 did not lead to induction of differentiation. Several defects in the cytokine triggered Jak/Stat signal pathway were striking in these lines. They expressed a decreased or undetectable amount of at least one of the components of the specific cytokine receptor complexes. Three lines contained a constitutively activated Jak/Stat signal cascade and in two of them, lines C and BMC-63, this cascade was inducible by treatment with IL-6, despite of a very low density of IL-6-receptors. Apart from the cytokine receptors, additional components of the Jak/Stat signal cascade were altered in these lines. Expression and activation of the transcription factor Stat5a and the tyrosine kinase Jak2 were markedly decreased compared to M1 cells, suggesting a role of activated Stat5a in the induction of differentiation. These results demonstrate a direct correlation between alterations in the Jak/Stat signal pathway and the inability to differentiate after cytokine treatment of myeloid leukaemia cells.

Members of the family of IL-6-type cytokines activate Stat5a in various cell types.

Interleukin-6 (IL-6)-type cytokines activate transcription factors Stat1 and Stat3 (signal transducers and activators of transcription). Here we report that leukemia inhibitory factor (LIF) and IL-6 activate Stat5a in M1 myeloid leukemia cells in addition. In murine embryonal stem (ES) cells stably transfected with an expression vector for Stat5a treatment with LIF resulted in tyrosine phosphorylation and DNA-binding of this transcription factor. Transfection of an expression construct for Stat5a in human hepatoma cells caused a dose-dependent increase in LIF-triggered transcriptional activity. Our data demonstrate that Stat5a is activated by IL-6-type cytokines and can mediate transcriptional activity in addition to Stat1 and Stat3.