Prognostic biomarkers for HNSCC using quantitative real-time PCR and microarray analysis: ß-tubulin isotypes and the p53 interactome.

In 2014, more than 40,000 people in the United States will be diagnosed with head and neck squamous cell cancer (HNSCC) and nearly 8400 people will die of the disease (www.cancer.org/acs/groups). Little is known regarding molecular targets that might lead to better therapies and improved outcomes for these patients. The incorporation of taxanes into the standard cisplatin/5-fluouracil initial chemotherapy for HNSCC has been associated with improved response rate and survival. Taxanes target the ß-subunit of the tubulin heterodimers, the major protein in microtubules, and halt cell division at G2/M phase. Both laboratory and clinical research suggest a link between ß-tubulin expression and cancer patient survival, indicating that patterns of expression for ß-tubulin isotypes along with activity of tumor suppressors such as p53 or micro-RNAs could be useful prognostic biomarkers and could suggest therapeutic targets. © 2014 Wiley Periodicals, Inc.

Regulation of tubulin expression by micro-RNAs: implications for drug resistance.

In this chapter, we provide an overview of methods for studying micro-RNA regulation of tubulin isotypes. In clinical studies, ß-tubulin isotypes were found to be biomarkers for tumor formation. In addition, because changes in the levels of specific ß-tubulin isotypes alter the stability of microtubules in mitotic spindles in vitro, it has been hypothesized that changes in microtubule protein levels could contribute to chemotherapy resistance. Over the past 15 years, micro-RNAs have been shown to target mRNAs in signaling pathways involved in tumor suppression, as well as tumorigenesis. Investigating micro-RNA regulation of tubulin isotypes will shed light on the mechanisms underlying the processes that implicate tubulin isotypes as biomarkers for aggressive tumors or chemotherapy resistance. The methods discussed in this chapter include the use of micro-RNA superarrays, next-generation sequencing, real-time PCR experiments, upregulation of micro-RNAs, and immunoprecipitation of RNA-induced silencing complex. We will show examples of data collected using these methods and how these data contribute to understanding paclitaxel resistance.

Coordinated regulation of ß-tubulin isotypes and epithelial-to-mesenchymal transition protein ZEB1 in breast cancer cells.

The regulation of ß-tubulin isotypes, the primary targets for antimitotic chemotherapeutic drugs like taxanes, has implications for drug response and drug resistance. Over the past 15 years, micro-RNAs have been studied widely as regulators of mRNA levels. For example, the tumor suppressor miR-200c was shown in cell culture to target mesenchymal genes, including ZEB1 [ Cochrane ( 2009 ) Mol. Cancer Ther. 8 ( 5 ), 1055 - 1066 ]. In that work, exogenous miR-200c was also shown to reduce ß-tubulin class III, one of its predicted targets. Furthermore, decreased miR-200c and increased ß-tubulin class III were associated with poor outcomes for ovarian cancer patients [ Cittelly , D.M. ( 2012 ) Mol. Cancer Ther. 11 ( 12 ), 2556 - 2565 ]. Because miR-200c targets the epithelial-to-mesenchymal inducer ZEB1, we wanted to know whether changes in ZEB1 parallel ß-tubulin isotype changes, implicating ß-tubulin isotypes in ZEB1-associated cell survival pathways. We found coordinated positive feedback regulation of mRNA for ZEB1 and ß-tubulin isotype classes I, III, and IVB in MDA-MB-231 breast cancer cells, commonly used as a model for triple-negative breast cancers. Low levels of paclitaxel (40 nM) were found to significantly reduce mRNA levels for these tubulin genes along with a 2-3-fold increase in miR-200c. ZEB1 silencing also reduced ß-tubulin isotype classes I, III, and IVB mRNA, whereas upregulation of ZEB1 was associated with increases in these isotype classes. Our work indicates that paclitaxel-induced reduction of ZEB1 and ß-tubulin isotypes are, in part, due to increased activity of miR-200c. These results suggest that in aggressive breast cancers, as modeled by MDA-MB-231 cells, ß-tubulin class III is a biomarker for cell survival mediated through ZEB1-induced tumor progression pathways.