Marjorie Liu

Microtubule alterations and mutations induced by desoxyepothilone B: implications for drug-target interactions.

Epothilones, like paclitaxel, bind to beta-tubulin and stabilize microtubules. We selected a series of four leukemia sublines that display increasing levels of resistance to the epothilone analog desoxyepothilone B (dEpoB). The dEpoB cells selected in 30-140 nM were approximately 15-fold cross-resistant to paclitaxel, while 300 nM selected cells were 467-fold resistant to this agent. The dEpoB-selected cells are hypersensitive to microtubule destabilizing agents, and express increased levels of class III beta-tubulin and MAP4. A novel class I beta-tubulin mutation, A231T, that affects microtubule stability but does not alter paclitaxel binding, was identified. The 300 nM selected cells acquired a second mutation, Q292E, situated near the M loop of class I beta-tubulin. These cells fail to undergo drug-induced tubulin polymerization due to dramatically reduced drug binding. The dEpoB-resistant leukemia cells provide novel insights into microtubule dynamics and, in particular, drug-target interactions.

Integrative analysis of RUNX1 downstream pathways and target genes

BackgroundThe RUNX1 transcription factor gene is frequently mutated in sporadic myeloid and lymphoid leukemia through translocation, point mutation or amplification. It is also responsible for a familial platelet disorder with predisposition to acute myeloid leukemia (FPD-AML). The disruption of the largely unknown biological pathways controlled by RUNX1 is likely to be responsible for the development of leukemia. We have used multiple microarray platforms and bioinformatic techniques to help identify these biological pathways to aid in the understanding of why RUNX1 mutations lead to leukemia.ResultsHere we report genes regulated either directly or indirectly by RUNX1 based on the study of gene expression profiles generated from 3 different human and mouse platforms. The platforms used were global gene expression profiling of: 1) cell lines with RUNX1 mutations from FPD-AML patients, 2) over-expression of RUNX1 and CBFβ, and 3) Runx1 knockout mouse embryos using either cDNA or Affymetrix microarrays. We observe that our datasets (lists of differentially expressed genes) significantly correlate with published microarray data from sporadic AML patients with mutations in either RUNX1 or its cofactor, CBFβ. A number of biological processes were identified among the differentially expressed genes and functional assays suggest that heterozygous RUNX1 point mutations in patients with FPD-AML impair cell proliferation, microtubule dynamics and possibly genetic stability. In addition, analysis of the regulatory regions of the differentially expressed genes has for the first time systematically identified numerous potential novel RUNX1 target genes.ConclusionThis work is the first large-scale study attempting to identify the genetic networks regulated by RUNX1, a master regulator in the development of the hematopoietic system and leukemia. The biological pathways and target genes controlled by RUNX1 will have considerable importance in disease progression in both familial and sporadic leukemia as well as therapeutic implications.

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.

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.

Evolution of resistance to Aurora kinase B inhibitors in leukaemia cells.

Aurora kinase inhibitors are new mitosis-targeting drugs currently in clinical trials for the treatment of haematological and solid malignancies. However, knowledge of the molecular factors that influence sensitivity and resistance remains limited. Herein, we developed and characterised an in vitro leukaemia model of resistance to the Aurora B inhibitor ZM447439. Human T-cell acute lymphoblastic leukaemia cells, CCRF-CEM, were selected for resistance in 4 µM ZM447439. CEM/AKB4 cells showed no cross-resistance to tubulin-targeted and DNA-damaging agents, but were hypersensitive to an Aurora kinase A inhibitor. Sequencing revealed a mutation in the Aurora B kinase domain corresponding to a G160E amino acid substitution. Molecular modelling of drug binding in Aurora B containing this mutation suggested that resistance is mediated by the glutamate substitution preventing formation of an active drug-binding motif. Progression of resistance in the more highly selected CEM/AKB8 and CEM/AKB16 cells, derived sequentially from CEM/AKB4 in 8 and 16 µM ZM447439 respectively, was mediated by additional defects. These defects were independent of Aurora B and multi-drug resistance pathways and are associated with reduced apoptosis mostly likely due to reduced inhibition of the catalytic activity of aurora kinase B in the presence of drug. Our findings are important in the context of the use of these new targeted agents in treatment regimes against leukaemia and suggest resistance to therapy may arise through multiple independent mechanisms.

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.

Abstract A156: Mechanisms of resistance to Aurora kinase B inhibitors in leukemia: Development and characterization of in vitro models

Aurora kinase inhibitors are generating great interest as new mitosis targeting drugs in cancer chemotherapy. Whilst these new agents are progressing through clinical trials against both solid and haematological malignancies, knowledge of the molecular factors that influence sensitivity and resistance remains limited. Development of optimal treatment modalities and the design of next generation Aurora inhibitors requires an understanding of processes and pathways of drug sensitivity mechanisms. In this study we report the development and characterisation of an in vitro derived leukaemia model of resistance to the Aurora B inhibitor ZM447439. CCRF‐CEM cells were selected for resistance in 4uM ZM447439 and designated CEM/AKB4 cells. Cytotoxicity assays showed that CEM/AKB4 cells were 13.2 fold resistant to ZM44739 compared to parental CEM cells. Resistance was not associated with the multi‐drug resistance phenotype and CEM/AKB4 cells showed no cross‐resistance to a number of tubulin‐targeting mitotic poisons or DNA‐damaging agents. The CEM/AKB4 cells remained sensitive to the Aurora kinase A inhibitor, ENMD‐2076. Cell cycle analysis revealed that exposure of CEM cells to ZM447439 (0.4–4 uM) caused extensive cell cycle disruption and cell death. In contrast, the cell cycle profiles of CEM/AKB4 cells at the same concentrations of drug were barely altered. Full length sequencing of the Aurora B gene revealed a single point mutation in the kinase domain corresponding to a G160E amino acid substitution. Molecular modelling of drug binding in Aurora B containing this mutation suggested that resistance is mediated by the glutamate substitution preventing formation of an active binding motif for Aurora B inhibitors. Moreover molecular docking of an Aurora B inhibitor with a novel binding motif was unchanged in the mutant enzyme suggesting this drug may abrogate resistance. Aurora kinase B inhibitor resistant cells are a valuable tool for identifying resistance mechanisms and the design of new kinase inhibitors active against Aurora kinase B mutations. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A156.