Abstract 5026: βIII-Tubulin regulates expression of proteins involved in tumorigenesis and metastasis in non-small cell lung cancer
Authors: McCarroll Joshua A. , Gan Pei Pei , Liu Marjorie , Dwarte Tanya , Chang Melissa , Byrne Frances , Kavallaris Maria 
Published: February 2011

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 drugs1. We have also recently shown that βIII-tubulin plays a role in regulating tumor growth both in vitro and in vivo2. Importantly, high levels of βIII-tubulin are correlated with more aggressive and drug refractory tumors in the clinic3. 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/βIIISH4 and pRS/βIIISH59) or control (pRS/CtrlSH1 and pRS/CtrlSH2). 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/βIIISH4 and pRS/βIIISH59 and pRS/CtrlSH1 and pRS/CtrlSH2 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/βIIISH4 and 59 cells when compared to their controls (pRS/CtrlSH1 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/βIIISH4 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/βIIISH4 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/βIIISH4 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

2McCarroll et al. 100th Annual AACR meeting. A3337, 2009

3Seve &Dumontet. Lancet Oncol, 9:168-75, 2008

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 5026.