Specific β-Tubulin Isotypes Can Functionally Enhance or Diminish Epothilone B Sensitivity in Non-Small Cell Lung Cancer Cells

Abstract

Epothilones are a new class of microtubule stabilizing agents with promising preclinical and clinical activity. Their cellular target is β-tubulin and factors influencing intrinsic sensitivity to epothilones are not well understood. In this study, the functional significance of specific β-tubulin isotypes in intrinsic sensitivity to epothilone B was investigated using siRNA gene knockdown against βII-, βIII- or βIVb-tubulins in two independent non-small cell lung cancer (NSCLC) cell lines, NCI-H460 and Calu-6. Drug-treated clonogenic assays showed that sensitivity to epothilone B was not altered following knockdown of βII-tubulin in both NSCLC cell lines. In contrast, knockdown of βIII-tubulin significantly increased sensitivity to epothilone B. Interestingly, βIVb-tubulin knockdowns were significantly less sensitive to epothilone B, compared to mock- and control siRNA cells. Cell cycle analysis of βIII-tubulin knockdown cells showed a higher percentage of cell death with epothilone B concentrations as low as 0.5 nM. In contrast, βIVb-tubulin knockdown cells displayed a decrease in epothilone B-induced G2-M cell cycle accumulation compared to control siRNA cells. Importantly, βIII-tubulin knockdowns displayed a significant dose-dependent increase in the percentage of apoptotic cells upon treatment with epothilone B, as detected using caspase 3/7 activity and Annexin-V staining. Higher concentrations of epothilone B were required to induce apoptosis in the βIVb-tubulin knockdowns compared to control siRNA, highlighting a potential mechanism underlying decreased sensitivity to this agent. This study demonstrates that specific β-tubulin isotypes can influence sensitivity to epothilone B and may influence differential sensitivity to this promising new agent.

Delineating the Role of βIV-Tubulins in Pancreatic Cancer: βIVb-Tubulin Inhibition Sensitizes Pancreatic Cancer Cells to Vinca Alkaloids

“…Similarly, few studies have explored the role of βIVb-tubulin in cancer cells [18], [24], [25], [33]. Notably, we demonstrated that βIVb-tubulin knockdown has significant effects on PDA cell growth and chemosensitivity.…”

“…These results are not surprising given that βIVa-tubulin appears to make up very little of the total βIV-tubulin pool in PDA cells. To date, there are few reported studies examining βIV-tubulin in cancer [16], [17], [18], [24], [25], [26], [33]. Only four studies have correlated increased βIVa-tubulin expression with increased chemoresistance (to estramustine and paclitaxel) in prostate, ovarian and lung cancer cells [16], [17], [26], [30].…”

Tubulin‐β‐III overexpression by uterine serous carcinomas is a marker for poor overall survival after platinum/taxane chemotherapy and sensitivity to epothilones

“…45 Given that heightened sensitivity to paclitaxel in response to tubulin-β-III downregulation had not yet been demonstrated in USC cell lines, we used siRNA to reduce tubulin-β-III expression; consistent with previous studies, we witnessed improved in vitro sensitivity of both USC and OSC to paclitaxel by approximately 2-fold. Tubulin-β-III downregulation has been previously shown to improve sensitivity to multiple chemotherapeutic agents, 46 including epothilone B 47 by predisposing cells to apoptosis through mechanisms other than reduction in mitotic block induced by chemotherapeutic agent. 46–47 The results of our siRNA tubulin-β-III knockdown experiments in USC are in agreement with this view and suggest that tubulin-β-III may not only serve as a marker for sensitivity to epothilones but also as a mediator of multiple cell survival pathways.…”

TUBB3/βIII-Tubulin Acts through the PTEN/AKT Signaling Axis to Promote Tumorigenesis and Anoikis Resistance in Non–Small Cell Lung Cancer

“…Clinical studies show that high bIII-tubulin expression correlates with chemoresistance and poor survival in different tumor types, including breast, ovarian, gastric, and NSCLC (5,6). Previously, we demonstrated a functional role for bIIItubulin in regulating chemosensitivity in NSCLC using RNA interference to silence bIII-tubulin expression in NSCLC cells (7)(8)(9)(10). Knockdown of bIII-tubulin increased sensitivity via an increase in apoptosis to chemotherapeutic agents both in vitro and in vivo (7)(8)(9)(10).…”

“…Previously, we demonstrated a functional role for bIIItubulin in regulating chemosensitivity in NSCLC using RNA interference to silence bIII-tubulin expression in NSCLC cells (7)(8)(9)(10). Knockdown of bIII-tubulin increased sensitivity via an increase in apoptosis to chemotherapeutic agents both in vitro and in vivo (7)(8)(9)(10). However, despite evidence to link bIII-tubulin expression to chemotherapy drug sensitivity, its roles in the tumorigenic and metastatic potential of NSCLC are unknown.…”