First line therapy for non-small cell lung carcinoma (NSCLC) commonly includes combination therapy with a tubulinbinding agent (TBA) and a DNA-damaging agent. TBAs suppress microtubule dynamics by binding to the B-tubulin subunit of A/B-tubulin, inducing mitotic arrest and apoptosis. Up-regulation of class III B-tubulin (BIII-tubulin) has been implicated in clinical resistance in NSCLC, ovarian and breast tumors treated in combination with a TBA and DNAdamaging agent. To investigate the functional significance of BIII-tubulin in resistance to both these classes of agents, small interfering RNA (siRNA) was used to silence the expression of this isotype in two NSCLC cell lines, NCI-H460 and Calu-6. Reverse transcription-PCR and immunoblotting showed that BIII-siRNA potently inhibited the expression of BIII-tubulin, without affecting the expression of other major B-tubulin isotypes. Clonogenic assays showed that BIII-siRNA cells were significantly more sensitive to TBAs, paclitaxel, vincristine, and vinorelbine, and for the first time, DNA-damaging agents, cisplatin, doxorubicin, and etoposide compared with controls. Cell cycle analysis of H460 BIII-siRNA cells showed reduced accumulation at the G 2 -M boundary and an increase in the sub-G 1 population in response to TBA treatment compared with control cells. Importantly, BIII-siRNA cells displayed a significant dose-dependent increase in Annexin V staining when treated with either paclitaxel or cisplatin, compared with controls. These findings have revealed a novel role for BIII-tubulin in mediating response to both TBA and DNAdamaging agent therapy and may have important implications for improving the targeting and treatment of drug-refractory NSCLC.

Significance Nuclear envelope breakdown (NEB) leads to the exposure of nuclear structures to cytoplasmic activities. Greatwall is a kinase able to inhibit PP2A phosphatases that counteract Cdk-dependent phosphorylation required for mitosis. Here we show that Greatwall, an essential protein in mammals, is exported to the cytoplasm in a Cdk-dependent manner before NEB, thus protecting mitotic phosphates from phosphatase activity.

Advanced non-small cell lung cancer (NSCLC) has a dismal prognosis. βIII-Tubulin, a protein highly expressed in neuronal cells, is strongly associated with drug-refractory and aggressive NSCLC. To date, the role of this protein in in vivo drug resistance and tumorigenesis has not been determined. NSCLC cells stably expressing βIII-tubulin short hairpin RNA displayed reduced growth and increased chemotherapy sensitivity when compared with control clones. In concordance with these results, stable suppression of βIII-tubulin reduced the incidence and significantly delayed the growth of tumors in mice relative to controls. Our findings indicate that βIII-tubulin mediates not only drug sensitivity but also the incidence and progression of lung cancer. βIII-Tubulin is a cellular survival factor that, when suppressed, sensitizes cells to chemotherapy via enhanced apoptosis induction and decreased tumorigenesis. Findings establish that upregulation of a neuronal tubulin isotype is a key contributor to tumor progression and drug sensitivity in lung adenocarcinoma.

Overexpression of βIII-tubulin is associated with resistance to tubulin-binding agents (TBA) in a range of tumor types. We previously showed that small interfering RNA silencing of βIII-tubulin expression hypersensitized non-small cell lung cancer cells to TBAs. To determine whether βIII-tubulin mediates its effect on drug-induced mitotic arrest and cell death by differentially regulating microtubule behavior, the effects of βIII-tubulin knockdown on microtubule dynamics were analyzed in H460 non-small cell lung cancer cells stably expressing green fluorescent protein-βI-tubulin. Interphase cells were examined at three vincristine and paclitaxel concentrations that (a) inhibited cell proliferation, (b) induced 5% to 10% mitotic arrest, and (c) induced 30% to 40% mitotic arrest. In the absence of either drug, βIII-tubulin knockdown caused no significant change in microtubule dynamic instability. At 2 nmol/L vincristine (IC 50 ), overall microtubule dynamicity was significantly suppressed in βIII-tubulin knockdowns (−31.2%) compared with controls (−6.5%). Similar results were obtained with paclitaxel, suggesting that knockdown of βIII-tubulin induces hypersensitivity by enhancing stabilization of microtubule dynamics at low drug concentrations. At higher drug concentrations (≥40 nmol/L vincristine; ≥20 nmol/L paclitaxel), βIII-tubulin knockdown resulted in significantly reduced suppressive effects on microtubule dynamicity with little or no further increase in mitotic arrest, compared with control cells. Importantly, apoptosis was markedly increased by βIII-tubulin knockdown independent of further suppression of microtubule dynamics and mitotic arrest. These results show that βIII-tubulin knockdown enhances the effectiveness of TBAs through two mechanisms: suppression of microtubule dynamics at low drug concentrations and a mitosis-independent mechanism of cell death at higher drug concentrations. Mol Cancer Ther; 9(5); 1339-48. ©2010 AACR.