β-Tubulin mutational analysis: tantalizing findings

Abstract

Resistance to taxanes could be due to alterations in the intracellular target, tubulin. Such alterations can include decreased tubulin, point mutations, altered expression of β-tubulin isotypes and acetylation of α-tubulin. Acquired β-tubulin mutations in human cancer cell lines confer resistance to taxanes and epothilones by impairing their binding to microtubules [ [1] Giannakakou P. Gussio R. Nogales E. et al. A common pharmacophore for epothilone and taxanes: molecular basis for drug resistance conferred by tubulin mutations in human cancer cells. Proc. Natl. Acad. Sci. USA. 2000; 97: 2904-2909 Crossref PubMed Scopus (468) Google Scholar ]. We have read with great interest the comprehensive mutational analysis of the β-tubulin gene by Tsurutani et al. [ [2] Tsurutani J. Komiya T. Uejima H. et al. Mutational analysis of the β-tubulin gene in lung cancer. Lung Cancer. 2002; 35: 11-16 Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar ], whose findings mirror those recently reported by Kelley et al. [ [3] Kelley M.J. Li S. Harpole D.H. Genetic analysis of the β-tubulin gene, TUBB, in non-small-cell lung-cancer. J. Natl. Cancer Inst. 2001; 93: 1886-1888 Crossref PubMed Scopus (75) Google Scholar ]. Both studies point out that when the originally described exonic primers for β-tubulin exon 4 [ [4] Monzó M. Rosell R. Sánchez J.J. et al. Paclitaxel resistance in non-small-cell lung cancer associated with beta-tubulin gene mutations. J. Clin. Oncol. 1999; 17: 1786-1793 PubMed Google Scholar ] were used, no real tubulin mutations were detected. Instead, multiple non-specific nucleotide sequences [ [2] Tsurutani J. Komiya T. Uejima H. et al. Mutational analysis of the β-tubulin gene in lung cancer. Lung Cancer. 2002; 35: 11-16 Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar ] or variants [ [3] Kelley M.J. Li S. Harpole D.H. Genetic analysis of the β-tubulin gene, TUBB, in non-small-cell lung-cancer. J. Natl. Cancer Inst. 2001; 93: 1886-1888 Crossref PubMed Scopus (75) Google Scholar ] were identified in 17 non-small-cell lung cancer (NSCLC) tumor samples and several lung cancer cell lines [ [2] Tsurutani J. Komiya T. Uejima H. et al. Mutational analysis of the β-tubulin gene in lung cancer. Lung Cancer. 2002; 35: 11-16 Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar ] and in 25 NSCLC cell lines and 20 NSCLC tumor samples [ [3] Kelley M.J. Li S. Harpole D.H. Genetic analysis of the β-tubulin gene, TUBB, in non-small-cell lung-cancer. J. Natl. Cancer Inst. 2001; 93: 1886-1888 Crossref PubMed Scopus (75) Google Scholar ]. In spite of the fact that many of these nucleotide substitutions lead to amino acid changes, mainly missense mutations [ [3] Kelley M.J. Li S. Harpole D.H. Genetic analysis of the β-tubulin gene, TUBB, in non-small-cell lung-cancer. J. Natl. Cancer Inst. 2001; 93: 1886-1888 Crossref PubMed Scopus (75) Google Scholar ], they were interpreted as pseudogenes. However, with cDNA [ [2] Tsurutani J. Komiya T. Uejima H. et al. Mutational analysis of the β-tubulin gene in lung cancer. Lung Cancer. 2002; 35: 11-16 Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar ] or intronic primers [ [3] Kelley M.J. Li S. Harpole D.H. Genetic analysis of the β-tubulin gene, TUBB, in non-small-cell lung-cancer. J. Natl. Cancer Inst. 2001; 93: 1886-1888 Crossref PubMed Scopus (75) Google Scholar ], such alterations were not visible, and only silent mutations were observed in 7/20 cell lines and in 4/22 lung tumors [ [2] Tsurutani J. Komiya T. Uejima H. et al. Mutational analysis of the β-tubulin gene in lung cancer. Lung Cancer. 2002; 35: 11-16 Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar ]. In the Kelley et al. [ [3] Kelley M.J. Li S. Harpole D.H. Genetic analysis of the β-tubulin gene, TUBB, in non-small-cell lung-cancer. J. Natl. Cancer Inst. 2001; 93: 1886-1888 Crossref PubMed Scopus (75) Google Scholar ] study, only 2/25 tumor cell lines were described as having either silent mutations or polymorphisms. Interestingly, the substitution at codon 217 found by Kelley et al. has been identified in several studies and was originally reported in paclitaxel-resistant Chinese hamster ovarian cell lines [ [5] González-Garay M.L. Chang L. Blade K. Menick D.R. Cabral F. A β-tubulin leucine cluster involved in microtubule assembly and paclitaxel resistance. J. Biol. Chem. 1999; 274: 23875-23882 Crossref PubMed Scopus (136) Google Scholar ]. The Leu 217 silent polymorphism has also been described in another study [ [6] Sale S. Sung R. Shen P. et al. Conservation of the class β-tubulin gene in human populations and lack of mutations in lung cancers and paclitaxel-resistant ovarian cancers. Mol. Cancer Ther. 2002; 1: 215-225 PubMed Google Scholar ], where no β-tubulin mutations were identified in ovarian or lung cancer tumor samples and cell lines with denaturing high pressure liquid chromatography. Recently however, in the EpoB-resistant A549 human NSCLC line, a mutation was noted at codon 292 [ [7] He L. Huang Yang Ch.-P. Horwitz S.B. Mutations in β-tubulin map to domains involved in regulation of microtubule stability in epothilone-resistant cell lines. Mol. Cancer Ther. 2001; 1: 3-10 PubMed Google Scholar ]. In our recent experience, as well as in that of other investigators (Gumerlock, Gandara, unpublished data), no tubulin mutations were found in either tumor or serum DNA from NSCLC patients, regardless of disease stage. These mutational analysis were performed using a forward intronic primer at position 2901, yielding a PCR product of 1200 bp. With this same methodology, no tubulin mutations have been observed in any patients included in a prospective ongoing tailored chemotherapy trial. At the time we designed the original primers in 1996 [ [4] Monzó M. Rosell R. Sánchez J.J. et al. Paclitaxel resistance in non-small-cell lung cancer associated with beta-tubulin gene mutations. J. Clin. Oncol. 1999; 17: 1786-1793 PubMed Google Scholar ], less information on tubulin genes and pseudogenes was available, and many of the originally described mutations may have been pseudogenes arising by retrotransposition. In a few instances, retrotransposition is maintained as a functional intronless gene, as is the case with ΨPTEN [ [8] Fujii G.H. Morimoto A.M. Berson A.E. Bolen J.B. Transcriptional analysis of the PTEN/MMAC1 pseudogene, ψPTEN. Oncogene. 1999; 18: 1765-1769 Crossref PubMed Scopus (76) Google Scholar ]. Pseudogene transcription may also modulate normal gene activity, and this phenomenon may well be the most plausible explanation for the survival differences found in our original study [ [4] Monzó M. Rosell R. Sánchez J.J. et al. Paclitaxel resistance in non-small-cell lung cancer associated with beta-tubulin gene mutations. J. Clin. Oncol. 1999; 17: 1786-1793 PubMed Google Scholar ]. Further research is being pursued to elucidate this issue. The contradictory results between mutations found in cell lines and tumor tissues have no easy explanation. Cancer therapy resistance is multifactorial, including the DNA repair machinery, checkpoint kinase Chk1 [ [9] Yarden R.I. Pardo-Reoyo S. Sgagias M. Cowan K.H. Brody L.C. BRCA1 regulates the G2/M checkpoint by activating Chk1 kinase upon DNA damage. Nature Genet. 2002; 30: 285-289 Crossref PubMed Scopus (396) Google Scholar ]. In addition, the loss of p53 function makes β-tubulin mutations irreversible [ [10] Giannakakou P. Poy G. Zhan Z. Knutsen T. Blagosklonny M.V. Fojo T. Paclitaxel selects for mutant or pseudo-null p53 in drug resistance associated with tubulin mutations in human cancer. Oncogene. 2000; 19: 3078-3085 Crossref PubMed Scopus (64) Google Scholar ]. Current lines of tubulin research are focussing on clarifying whether up-regulation of certain β-tubulin isotypes could become a pretherapeutic determinant of the response to microtubule-damaging agents.