Pei Pei Gan

Class III β-tubulin mediates sensitivity to chemotherapeutic drugs in non-small cell lung cancer

First line therapy for non–small cell lung carcinoma (NSCLC) commonly includes combination therapy with a tubulin-binding agent (TBA) and a DNA-damaging agent. TBAs suppress microtubule dynamics by binding to the β-tubulin subunit of α/β-tubulin, inducing mitotic arrest and apoptosis. Up-regulation of class III β-tubulin (βIII-tubulin) has been implicated in clinical resistance in NSCLC, ovarian and breast tumors treated in combination with a TBA and DNA-damaging agent. To investigate the functional significance of βIII-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 βIII-siRNA potently inhibited the expression of βIII-tubulin, without affecting the expression of other major β-tubulin isotypes. Clonogenic assays showed that βIII-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 βIII-siRNA cells showed reduced accumulation at the G2-M boundary and an increase in the sub-G1 population in response to TBA treatment compared with control cells. Importantly, βIII-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 βIII-tubulin in mediating response to both TBA and DNA-damaging agent therapy and may have important implications for improving the targeting and treatment of drug-refractory NSCLC. [Cancer Res 2007;67(19):9356–63]

βIII-Tubulin Is a Multifunctional Protein Involved in Drug Sensitivity and Tumorigenesis in Non–Small Cell Lung Cancer

Advanced non-small cell lung cancer (NSCLC) has a dismal prognosis. betaIII-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 betaIII-tubulin short hairpin RNA displayed reduced growth and increased chemotherapy sensitivity when compared with control clones. In concordance with these results, stable suppression of betaIII-tubulin reduced the incidence and significantly delayed the growth of tumors in mice relative to controls. Our findings indicate that betaIII-tubulin mediates not only drug sensitivity but also the incidence and progression of lung cancer. betaIII-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.

Microtubule Dynamics, Mitotic Arrest, and Apoptosis: Drug-Induced Differential Effects of βIII-Tubulin

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 (IC50), 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.

Tubulin-Targeted Drug Action: Functional Significance of Class II and Class IVb β-Tubulin in Vinca Alkaloid Sensitivity

Aberrant expression of beta-tubulin isotypes is frequently described in tumor tissues and tubulin-binding agent (TBA)-resistant cell lines. There is limited understanding of the role of specific beta-tubulin isotypes in cellular sensitivity to TBAs, and to gain insights into the functional role of betaII- and betaIVb-tubulin, we examined these isotypes in lung cancer cell lines NCI-H460 (H460) and Calu-6. Drug-treated clonogenic assays revealed that small interfering RNA-mediated knockdown of either betaII- or betaIVb-tubulin hypersensitized the lung cancer cell lines to Vinca alkaloids, with the effects more pronounced following betaIVb-tubulin knockdown. In contrast, there was no change in paclitaxel sensitivity following knockdown of either isotype. Cell cycle analysis revealed a greater propensity for the betaII- and betaIVb-tubulin knockdown cells to undergo G2-M cell cycle block following 5 nmol/L vincristine treatment, with the betaIVb knockdown cells being more sensitive than the betaII-tubulin knockdown cells compared with control. In contrast to betaII-tubulin knockdown, betaIVb-tubulin knockdown cells showed a significant increase in the sub-G1 population (cell death) following treatment with both 5 and 40 nmol/L of vincristine compared with controls. Importantly, betaIVb-tubulin knockdown in H460 cells caused a significant dose-dependent increase in Annexin V staining in response to vincristine but not paclitaxel. Therefore, increased sensitivity to induction of apoptosis is one mechanism underlying the Vinca alkaloid hypersensitivity. This study provides direct evidence that betaII- or betaIVb-tubulins have functionally distinct roles and expression of these isotypes may serve as strong predictors of Vinca alkaloid response and resistance.

Specific β-Tubulin Isotypes Can Functionally Enhance or Diminish Epothilone B Sensitivity in Non-Small Cell Lung Cancer Cells

Epothilones are a new class of microtubule stabilizing agents with promising preclinical and clinical activity. Their cellular target is β-tubulin and factors influencing intrinsic sensitivity to epothilones are not well understood. In this study, the functional significance of specific β-tubulin isotypes in intrinsic sensitivity to epothilone B was investigated using siRNA gene knockdown against βII-, βIII- or βIVb-tubulins in two independent non-small cell lung cancer (NSCLC) cell lines, NCI-H460 and Calu-6. Drug-treated clonogenic assays showed that sensitivity to epothilone B was not altered following knockdown of βII-tubulin in both NSCLC cell lines. In contrast, knockdown of βIII-tubulin significantly increased sensitivity to epothilone B. Interestingly, βIVb-tubulin knockdowns were significantly less sensitive to epothilone B, compared to mock- and control siRNA cells. Cell cycle analysis of βIII-tubulin knockdown cells showed a higher percentage of cell death with epothilone B concentrations as low as 0.5 nM. In contrast, βIVb-tubulin knockdown cells displayed a decrease in epothilone B-induced G2-M cell cycle accumulation compared to control siRNA cells. Importantly, βIII-tubulin knockdowns displayed a significant dose-dependent increase in the percentage of apoptotic cells upon treatment with epothilone B, as detected using caspase 3/7 activity and Annexin-V staining. Higher concentrations of epothilone B were required to induce apoptosis in the βIVb-tubulin knockdowns compared to control siRNA, highlighting a potential mechanism underlying decreased sensitivity to this agent. This study demonstrates that specific β-tubulin isotypes can influence sensitivity to epothilone B and may influence differential sensitivity to this promising new agent.

TUBB3/βIII-tubulin acts through the PTEN/AKT signaling axis to promote tumorigenesis and anoikis resistance in non-small cell lung cancer.

βIII-tubulin (encoded by TUBB3) expression is associated with therapeutic resistance and aggressive disease in non-small cell lung cancer (NSCLC), but the basis for its pathogenic influence is not understood. Functional and differential proteomics revealed that βIII-tubulin regulates expression of proteins associated with malignant growth and metastases. In particular, the adhesion-associated tumor suppressor maspin was differentially regulated by βIII-tubulin. Functionally, βIII-tubulin suppression altered cell morphology, reduced tumor spheroid outgrowth, and increased sensitivity to anoikis. Mechanistically, the PTEN/AKT signaling axis was defined as a critical pathway regulated by βIII-tubulin in NSCLC cells. βIII-Tubulin blockage in vivo reduced tumor incidence and growth. Overall, our findings revealed how βIII-tubulin influences tumor growth in NSCLC, defining new biologic functions and mechanism of action of βIII-tubulin in tumorigenesis.

Abstract 2076: βIII-tubulin is required for the tumorigenic phenotype and resistance to anoikis via the PTEN/AKT signaling axis in non-small cell lung cancer

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA BACKGROUND: Non-small cell lung cancer (NSCLC) has a dismal prognosis and remains the most common cause of cancer death worldwide. Expression of βIII-tubulin, encoded by the TUBB3 gene, is associated with clinical resistance and aggressive disease in NSCLC1. Herein, we interrogated the mechanistic role of βIII-tubulin in regulating the tumorigenic potential of NSCLC. METHODS: Functional studies involved independent clones of NSCLC H460 cells stably expressing shRNA targeting βIII-tubulin, H460 controls stably expressing (non-functional) shRNA and βIII-tubulin rescue clones established in our laboratory2. Differential proteomics was conducted using fluorescence based 2D-DIGE and mass spectrometry. Gene and protein expression performed by qRT-PCR and western blotting respectively. To assess effects on tumor growth and incidence we used metastatic (tail vein) and subcutaneous models of NSCLC. Tumors monitored by CT or Xenogen imaging. RESULTS: Functional and differential proteomics revealed that βIII-tubulin regulates expression of tumor growth- and metastases-associated proteins. In particular, the tumor suppressor maspin, associated with adhesion and metastasis, was differentially regulated by βIII-tubulin. Functionally, βIII-tubulin suppression led to altered cell morphology, increased cell adhesion and increased sensitivity to anoikis. Mechanistically, we identified PTEN and AKT kinase as a key signaling axis mediating anoikis and regulated by βIII-tubulin levels in NSCLC cells. Finally, βIII-tubulin suppression was shown to reduce NSCLC tumor growth and incidence in vivo. Collectively, these data identified βIII-tubulin as a regulator of tumor growth and metastasis through regulation of PTEN and AKT signaling. We conclude that suppressing βIII-tubulin may reduce tumor growth in NSCLC. SIGNIFICANCE: This is the first study to show that silencing βIII-tubulin alters the expression of proteins involved in promoting tumorigenicity and increases sensitivity to anoikis, leading to reduced tumor incidence. Targeting βIII-tubulin could be a promising strategy for inhibiting tumor growth and metastasis in NSCLC. 1 Kavallaris, M. Nature Rev Cancer, 10:194-204, 2010 2 McCarroll et al. Cancer Res 70 :4995-5003, 2010 Citation Format: Joshua A. McCarroll, Pei Pei Gan, Rafael B. Erlich, Marjorie Liu, Tanya Dwarte, Mia C. Akerfeldt, Melissa Chang, Michael S. Shum, Frances Byrne, Maria Kavallaris. βIII-tubulin is required for the tumorigenic phenotype and resistance to anoikis via the PTEN/AKT signaling axis in non-small cell lung cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2076. doi:10.1158/1538-7445.AM2014-2076

Abstract 5026: βIII-Tubulin regulates expression of proteins involved in tumorigenesis and metastasis in non-small cell lung cancer

Survival rates for advanced non-small cell lung cancer (NSCLC) remain dismal. Recently, we showed that silencing the microtubule protein βIII-tubulin using siRNA sensitizes NSCLC cells to chemotherapy drugs 1 . We have also recently shown that βIII-tubulin plays a role in regulating tumor growth both in vitro and in vivo 2 . Importantly, high levels of βIII-tubulin are correlated with more aggressive and drug refractory tumors in the clinic 3 . To date, the broader role of βIII-tubulin in NSCLC has not been determined and the aims of this study were to examine the role of βIII-tubulin on the expression of proteins involved in regulating tumorigenesis and metastasis. Methods: Using a functional and differential proteomics approach we examined NSCLC clones stably expressing shRNA against βIII-tubulin (pRS/βIII SH4 and pRS/βIII SH59 ) or control (pRS/Ctrl SH1 and pRS/Ctrl SH2 ). Cytosolic and nuclear protein fractions were prepared and 2-D DIGE was performed over broad (pI 4-7) and narrow (pI 4.5-5.5) pI ranges. Differences in protein expression between pRS/βIII SH4 and pRS/βIII SH59 and pRS/Ctrl SH1 and pRS/Ctrl SH2 cells were assessed using Decyder software. Protein spots of interest were excised and identified by mass spectrometry. Those identified as being significantly altered were then validated by western blot. Results: Eleven out of a total of 963 proteins (1.1%; pI 4-7) and 53 out of 753 proteins (7%; pI 4.5-5.5) were found to be significantly altered in the cytoplasmic fractions of pRS/βIII SH4 and 59 cells when compared to their controls (pRS/Ctrl SH1 and SH2 ). In addition, 33 out of 1331 proteins (2.5%; pI 4-7) and 42 out of 502 proteins (8.4%; pI 4.5-5.5) were significantly altered in the nuclear fractions of pRS/βIII SH4 and SH59 cells compared to controls. Importantly, a number of proteins which are involved in regulating tumor growth and metastasis were identified as being differentially expressed in the βIII-tubulin knockdown cells. A significant decrease (greater than 2 fold) in heat shock protein 60 expression (promotes metastasis) was observed in the pRS/βIII SH4 and SH59 nuclear fractions when compared to controls. Furthermore, a greater than 6 fold increase in the tumor suppressor protein Tropomyosin 1 and a greater than 2 fold increase in the serpin B5 precursor protein Maspin (inhibitor of metastasis) was identified in the pRS/βIII SH4 and SH59 cytoplasmic fractions when compared to controls. These changes were confirmed by western blotting. Conclusions: This is the first study to show that silencing βIII-tubulin significantly alters the expression of proteins involved in regulating tumorigenesis and metastasis in lung cancer. Targeting βIII-tubulin could be a promising strategy for inhibiting tumor growth and metastasis in lung cancer. 1 Gan et al. Cancer Res, 67:9356-63, 2007 2 McCarroll et al. 100 th Annual AACR meeting. A3337, 2009 3 Seve D 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5026.