Non-small cell lung cancer (NSCLC) is the most common and deadliest form of lung cancer. Despite aggressive treatment the survival rates remain dismal. High levels of the microtubule protein, βIII-tubulin (TUBB3), correlate with drug resistant and aggressive tumours in the clinic. Recently, we showed that silencing TUBB3 using RNA interference sensitises NSCLC cells to chemotherapy and plays a role in regulating tumour growth. However, how TUBB3 is mediating these effects in NSCLC is unknown. The aims of this study were to examine protein pathways regulated by TUBB3 using differential and functional proteomics.
Methods: NSCLC cells stably expressing shRNA against TUBB3 or control shRNA were developed. Cytosolic and nuclear protein fractions were prepared from these cells and 2-D DIGE was performed. Differences in protein expression between TUBB3 knockdown (KD) and control cellswere quantitatively assessed and protein spots of interest were excised and identified by mass spectrometry.
Results: Eleven out of a total of 963 proteins (pI 4-7) and 53/753 proteins (pI 4.5-5.5) were significantly altered in the cytoplasmic fractions of TUBB3 KD cells. In addition, 33 out of 1331 proteins (pI 4-7) and 42/502 proteins (pI 4.5-5.5) were significantly altered in the nuclear fractions of TUBB3 KD cells. Importantly, a number of proteins involved in regulating tumour growth and metastases were differentially expressed in the TUBB3 KD cells, including a significant increase in the tumour suppressor protein maspin. Differential expression of maspin was validated using western blotting and qPCR. Rescue of TUBB3 in the KD cells restored maspin levels to control levels, indicating a potential interaction between TUBB3 and maspin.
Conclusions: This is the first study to show that TUBB3 regulates the expression of proteins involved in tumourigenesis and metastasis in NSCLC. Targeting TUBB3 could be a promising strategy for inhibiting tumour growth and metastasis in lung cancer.