High TUBB3 Expression, an Independent Prognostic Marker in Patients with Early Non–Small Cell Lung Cancer Treated by Preoperative Chemotherapy, Is Regulated by K-Ras Signaling Pathway

Abstract

We assessed the prognostic and predictive value of b-tubulin III (TUBB3) expression, as determined by immunohistochemistry, in 412 non-small cell lung cancer (NSCLC) specimens from early-stage patients who received neoadjuvant chemotherapy (paclitaxel-or gemcitabine-based) in a phase III trial (IFCT-0002). We also correlated TUBB3 expression with K-Ras and EGF receptor (EGFR) mutations in a subset of 208 cryopreserved specimens. High TUBB3 protein expression was associated with nonsquamous cell carcinomas (P < 0.001) and K-Ras mutation (P < 0.001). The 127 (30.8%) TUBB3-negative patients derived more than 1 year of overall survival advantage, with more than 84 months median overall survival versus 71.7 months for TUBB3-positive patients [HR, 1.58; 95% confidence interval (CI), 1.11-2.25)]. This prognostic value was confirmed in multivariate analysis (adjusted HR for death, 1.51; 95% CI, 1.04-2.21; P ¼ 0.031) with a bootstrapping validation procedure. TUBB3 expression was associated with nonresponse to chemotherapy (adjusted HR, 1.31; 95% CI, 1.01-1.70; P ¼ 0.044) but had no predictive value (taxane vs. gemcitabine). Taking account of these clinical findings, we further investigated TUBB3 expression in isogenic human bronchial cell lines only differing by K-Ras gene status and assessed the effect of K-Ras short interfering RNA (siRNA) mediated depletion, cell hypoxia, or pharmacologic inhibitors of K-Ras downstream effectors, on TUBB3 protein cell content. siRNA K-Ras knockdown, inhibition of RAF/MEK (MAP-ERK kinase) and phosphoinositide 3-kinase (PI3K)/AKT signaling, and hypoxia were shown to downregulate TUBB3 expression in bronchial cells. This study is the first one to identify K-Ras mutations as determinant of TUBB3 expression, a chemoresistance marker. Our in vitro data deserve studies combining standard chemotherapy with anti-MEK or anti-PI3K drugs in patients with TUBB3-overexpressing tumors.

Cigarette smoke mediates epigenetic repression of miR-487b during pulmonary carcinogenesis

“…Overexpression of WNT5A increases proliferation, migration, invasion, and tumorigenicity of lung cancer cells in part via modulation of HOX family genes (D. Schrump et al, unpublished observations); elevated intratumoral levels of WNT5A correlate with increased neoangiogenesis, dissemination of disease, and decreased survival of lung cancer patients (47). MYC and KRAS activate a variety of pathways facilitating initiation and progression of lung cancer (24,48), and overexpression of these oncogenes correlates with treatment resistance and poor survival in lung cancer patients (49,50). Collectively, these observations strongly suggest that epigenetic repression of miR-487b enhances stemness phenotype of lung cancers and are consistent with our recent studies demonstrating that cigarette smoke upregulates ABCG2, increases pluripotent side population, and activates a variety of stem cell signaling pathways in lung cancer cells (51).…”

RASSF1A Suppresses the Invasion and Metastatic Potential of Human Non–Small Cell Lung Cancer Cells by Inhibiting YAP Activation through the GEF-H1/RhoB Pathway

“…12), tumorigenic epithelial cell lines BEAS-2B, cancer-derived cell lines A549 and H1975 from ATCC, and BEAS-2B-KRasV12 from Dr. Pradines (9,13) were grown in supplemented appropriate media (Supplementary Table S1), transfected at 30% confluence, using Lipofectamine RNAiMAX (Invitrogen) with siRNA, plasmid DNA, or control mimics (Dharmacon; Supplementary Tables S2 and S3). HBEC-3 and HBEC-3-KRasG12V were authenticated using standard karyotyping techniques as previously described (12). A549, H1975, BEAS-2B, were passaged for fewer than 6 months after receipt from ATCC.…”

Biomarkers for efficacy of adjuvant chemotherapy following complete resection in NSCLC stages I–IIIA

“…Microtubules are part of the cell's cytoskeleton and, therefore, crucial in cell division. Expression of TUBB3 may be seen in many different types of cancer cells [35]. High/positive expression of TUBB3 has been reported to be a prognostic marker for poor DFS and OS [14][15][16].…”