Xiaomeng Zhang and Qi Wang contributed equally to this article.
Cancer Cell Biology
Anti-apoptotic role of TWIST and its association with Akt pathway in mediating taxol resistance in nasopharyngeal carcinoma cells
Article first published online: 17 JAN 2007
Copyright © 2006 Wiley-Liss, Inc.
International Journal of Cancer
Volume 120, Issue 9, pages 1891–1898, 1 May 2007
How to Cite
Zhang, X., Wang, Q., Ling, M.-T., Wong, Y. C., Leung, S. C.L. and Wang, X. (2007), Anti-apoptotic role of TWIST and its association with Akt pathway in mediating taxol resistance in nasopharyngeal carcinoma cells. Int. J. Cancer, 120: 1891–1898. doi: 10.1002/ijc.22489
- Issue published online: 28 FEB 2007
- Article first published online: 17 JAN 2007
- Manuscript Accepted: 20 OCT 2006
- Manuscript Received: 26 JUN 2006
- Association for International Cancer Research, UK. Grant Number: HKU7478/03M
TWIST, a basic helix-loop-helix transcription factor, has been reported to be associated with development and progression of human cancer. Recently, over expression of TWIST is found in cancer patients with shorter survival and poor response to chemotherapy. Previously, we found that upregulation of TWIST was responsible for the development of acquired resistance to taxol in a nasopharyngeal carcinoma (NPC) cell line, HNE1-T3 (Wang et al., Oncogene, 2004;24:274). In this study, we investigated the underlying molecular mechanisms responsible for the TWIST-mediated taxol resistance. By comparison of the parental HNE1 and its derivative HNE1-T3 cell lines, we found that the resistance to taxol in HNE1-T3 cells was associated with suppression of taxol-induced apoptosis evidenced by decreased expression of Bak and Bax and increased Bcl-2, as well as inhibition of PARP and caspase cleavage and DNA ladder formation. However, there was no correlation between taxol sensitivity and alterations on G2/M cell cycle distribution, suggesting that the TWIST-induced taxol resistance is mediated through protection against apoptosis but not mitotic arrest. Analysis of additional 8 NPC cell lines showed that upregulation of TWIST was associated with resistance to microtubule disrupting agents, especially taxol, and inactivation of TWIST through small RNA interference led to increased sensitivity to taxol-induced cell death. Subsequent studies also demonstrated that the TWIST-mediated taxol resistance may be regulated through its positive involvement with the Akt pathway. Our findings suggest an underlying molecular mechanism responsible for the TWIST-mediated chemodrug resistance and suggest a target for overcoming taxol resistance in cancer cells. © 2007 Wiley-Liss, Inc.
TWIST, a highly conserved basic helix-loop-helix transcription factor, was first reported to play a key role in mesodormal, myoblast and osteoblast differentiation.1, 2, 3 Mutational inactivation of the TWIST gene is suggested to be responsible for the Saethre-Chotzen syndrome, an autosomal dominant disorder, characterized by premature fusion of the cranial sutures, skull deformations, limb abnormalities and facial dysmorphisims.4, 5, 6 Recently, TWIST has been suggested to play a positive role in the development and progression of human cancer. For example, over expression of TWIST is reported in human rhabdomyosarcoma,7 gastric carcinoma,8 melonoma,9 breast cancer,10, 11, 12, 13 prostate cancer14 and glioma.15 In addition, increased TWIST expression levels are associated with advance tumour stage and poor prognosis in several types of cancer.9, 14, 15, 16 More recently, increased TWIST in cancer cells has been shown to promote metastatic ability of breast cancer in in vivo animal models.12, 13 In osteosarcoma, upregulation of TWIST is found to be a predictor in patients with poor response to chemotherapy.17 Several in vitro studies also agree with these findings. For example, exogenous TWIST expression in mouse embryonic fibroblasts promotes soft agar colony formation,7 indicating its role in malignant transformation. Gene profiling analysis shows that upregulation of TWIST is associated with malignant transformation of melanoma and T cell lymphoma.9, 18 Transfection of TWIST in human cancer cells also increases invasion and metastatic abilities through promoting epithelial to mesenchymal transition.12, 13, 14, 15 Previously, we have found that TWIST gene amplification is associated with the development of acquired resistance to an anticancer drug, taxol, and ectopic expression of TWIST leads to resistance to microtubule disrupting agents.19 These results strongly suggest that TWIST may be a potential oncogene. Although the underlying mechanisms responsible for the positive role of TWIST in human cancer are not clear, several studies suggest that the ability of TWIST to promote cell survival rather than interfering with proliferation may account for its oncogenic effect. For example, the transformation effect of TWIST in mouse embryo fibroblasts is thought to be regulated through its ability to inhibit ARF/MDM2/p53 mediated apoptosis.7 In addition, while overexpression of TWIST in human breast and prostate cancer cells promotes cell survival and invasion ability, but it did not alter cell proliferation rate.13, 14 Previously, we found that the TWIST-mediated acquired taxol resistance is associated with suppression of Bax, indicating its role as an-antiapoptotic factor in mediating chemodrug resistance.19 The aim of this study was to continue our previous investigation to establish the molecular mechanisms responsible for the TWIST-induced taxol resistance, and to explore the possibility of using TWIST as a therapeutic target to overcome taxol resistance in nasopharyngeal carcinoma (NPC) cells. Using the 2 previously established cell lines, HNE1 and HNE1-T3, we found that the TWIST-mediated taxol resistance was mediated through its protection against apoptosis but not its effect on mitosis. We also observed a positive association between TWIST and Akt phosphorylation in HNE1-T3 cells, suggesting that the TWIST-mediated taxol resistance may be regulated through the Akt pathway. Studies on additional 8 NPC cell lines and Small RNA interference experiments demonstrated that upregulation of TWIST may be a predictor for taxol resistance and downregulation of TWIST may be a therapeutic target to overcome taxol resistance in cancer cells.
Material and methods
Cell culture and reagents
Eight human cancer cell lines derived from NPC, CNE1, CNE2, CNE3, HONE1, TW01, TW04, SUNE1, HNE1 and HK120 were maintained in RPMI 1640 (Life Technologies) supplemented with 2 mM L-glutamine and 5% (v/v) fetal calf serum at 37°C. The HNE1-T3 cell line was generated from HNE1 cell line by exposure to increasing concentrations of taxol for ∼12 months as described19 and maintained in drug-free RPMI 1640. PI3K and p38MAPK inhibitors/analogs, LY294002, Wortmannin, SB203580 and SB202474, were purchased from Calbiochem, CA, USA.
Measurement of cell growth
About 10,000 cells were plated in each well of 12-well plates. Number of cells was counted every day using a cell counter (Coulter, USA). Each data point was tested on triplicate wells and each experiment was repeated at least 3 times. Cell growth curves were drawn using the means of 3 experiments.
RT-PCR analysis of TWIST gene expression
Detailed experimental procedures and primers were described previously.19
Cell cycle analysis
Detailed experimental protocol has been published previously.14 Briefly, cells (5 × 105) were plated in 5% FCS culture medium and different concentrations of taxol were added for indicated time points. Both floating and attached cells were collected and fixed in ice-cold 70% ethanol. The cells were then washed with PBS and incubated with propidium iodide (50 μg/ml) and RNase (1 μg/ml) for 30 min. Flow cytometric analysis was performed on an EPICS profile analyzer and analyzed using the ModFit LT2.0 software (Coulter). Results represented average of 3 experiments.
Colony forming assay
Experimental details have been described elsewhere.21 Briefly, 200 cells were seeded per well in 6-well plates and 5 doses of taxol or vincristine were added 24 hr later for 10 days continuously. The cells were fixed in 70% ethanol and stained with 10% (v/v) Giemsa (MERCK, Damstadit, Germany). Colonies consisted of more than 50 cells were counted. Two wells were used for each dose and 2 wells treated with solvent were only used as controls. Colony forming ability after drug treatment was calculated as the ratio between the number of colonies in the treated wells and the untreated controls multiplied by 100. Results represented the average of 3 independent experiments.
3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay
Detailed methods was described previously.14 Briefly, 2,000 cells were seeded in 96-well plates and different concentrations of taxol were added respectively 24 hr later. Three wells were tested for each drug dose and cell viability was examined 24 hr after treatment. Results represented the OD ratio between the treated and untreated cells at the same indicated time points.
Detailed experimental procedures were described previously.14 Protein suspension from the whole-cell lysate (15∼30 μg) was loaded onto a sodium dodecylsulfate-polyacrylamide gel (SDS-PAGE) for electrophoresis and then transferred to a PVDF membrane (Amersham, Piscataway, NJ). The membrane was then incubated with primary antibodies for 1∼2 hr at room temperature against TWIST (H-81; Santa Cruz Biotechnology, Santa Cruz, CA), Bcl-2 (Santa Cruz Biotechnology), Bax (Cell Signaling Technology, Beverly, MA), Bak (Cell Signaling Technology), PARP(Cell Signaling Technology), Caspase-3 (Cell Signaling Technology), Akt (Cell Signaling Technology), p-Akt (Cell Signaling Technology), p-p38MAPK(Cell Signaling Technology), p38MAPK (Cell Signaling Technology) and Actin (Santa Cruz Biotechnology, CA) respectively. After washing with TBS-T, the membrane was incubated with a secondary antibody against mouse, rabbit or goat IgG accordingly and the signals were visualized using ECL plus Western blotting system (Amersham, Piscataway, NJ). Relative OD ratio was calculated by comparing to Actin from 3 experiments.
Detection of DNA ladder
Detailed experimental procedures were described previously.22
Generation of stable Si-TWIST transfectants
TWIST si-RNA vector was generated using the BLOCK IT™ RNAi Letiviral Expression System according to manufacture's instructions (Invitrogen, Carlsbad, CA). Briefly, the primers containing the short hairpin RNA sequence targeting the TWIST coding region was annealed and cloned into pENTR™/U6vector. Then the U6 RNAi cassette in pENTR™/U6vector was transferred to pLenti6/BLOCK-iT-DEST vector using Gateway LR Clonase II Enzyme Mix. The sequences of the si-TWIST primers were: si-TWIST-forward: 5′-CAC CGG ACA AGC TGA GCA AGA TTC ACG AAT GAA TCT TGC TCA GCT TGT CC-3′; si-TWIST-reverse: 5′-AAA AGG ACA AGC TGA GCA AGA TTC ATT CGT GAA TCT TGC TCA GCT TGT CC-3′. The control vector was generated using the same procedures as the si-RNA vector except that the short hairpin RNA sequence was replaced with nonsense sequences that are not homologous to the human genome. The sequences of the control primers were: si-Con-forward: CAC CGC GTA TTG CCT AGC ATT ACC GAA GTA ATG CTA GGC AAT ACG C; si-Con-reverse: AAA AGC GTA TTG CCT AGC ATT ACT TCG GTA ATG CTA GGC AAT ACG C. The resulting vectors were then transfected into the packaging cell line 293FT. The culture medium containing retroviruses were collected 48 hr later, mixed with polybrene (6 μg/ml), and then incubated with CNE1, CNE2 and HONE1, cells respectively. Positive si-TWIST clones were then selected in Blasticidin (2.5 μg/ml for CNE1 and CNE2, and 9 μg/ml for HONE1) and stable transfectants were isolated after ∼6 days drug selection. A pool of transfectants containing >20 positive clones were isolated from each cell line.
Association between cellular sensitivity and taxol-induced apoptosis
As discussed previously, TWIST has been suggested as an anti-apoptotic factor which protects cells against apoptosis in several cell types5, 7, 14 and our previous studies on these 2 lines showed that upregulation of TWIST in HNE1-T3 cells was associated with increased Bcl-2/Bax ratio,19 indicating a possible suppression of apoptosis pathway. To investigate if upregulation of TWIST in HNE1-T3 cells played a protective role against taxol-induced apoptosis, we studied the expression of Bcl-2, Bak, Bax as well as caspase 3 and PARP. As shown in Figure 1a, we found that the increased TWIST expression in HNE1-T3 cells was associated with lower levels of pro-apoptotic factors, Bak and Bax, and higher levels of Bcl-2 compared to HNE1 cells in response to taxol (5 ng/ml; 10 ng/ml) in a time dependent manner. In addition, there were no detectable levels of cleaved caspase 3 or PARP, an indication of activation of the apoptotic pathway, in HNE1-T3 cells after exposure to taxol. In contrast, increased cleaved caspase 3 and PARP was observed in a dose-dependent manner in HNE1 cells. These findings indicate that the high levels of TWIST in HNE1-T3 may provide a protection against taxol-induced apoptosis. To further confirm these results, we performed DNA ladder assay. As shown in Figure 1b, while there was no evidence of DNA fragmentation in HNE1-T3 cells under the conditions tested, DNA laddering was evident in HNE1 cells after exposure to the same doses of taxol (5, 10 ng/ml). These results suggest that high levels of TWIST may be able to function as an anti-apoptotic factor leading to resistance to taxol.
Role of Akt and p38MAP kinases in TWIST-mediated taxol resistance
Several signaling pathways have been suggested to play important roles in mediating anticancer drug-induced apoptosis in cancer cells. For example, activation of Akt is believed to protect taxol-induced apoptosis,23 while stimulation of the p38MAPK pathway leads to increased sensitivity to taxol.24 Next we investigated the involvement of Akt and p38MAPK in the TWIST-mediated taxol resistance. As shown in Figure 2a, activation of p-Akt and suppression of p-p38MAPK, evidenced by alterations of phosphorylated protein expression, were observed in HNE1-T3 cells compared to HNE1 cells. After exposure to taxol (5, 10 ng/ml), the differential p-Akt and p-p38MAPK expression between these 2 cell lines was more apparent in a dose dependent manner, suggesting the involvement of these 2 pathways in taxol response in these 2 cell lines. To further confirm this hypothesis, we then treated HNE1-T3 cells with 2 PI3K inhibitors, LY294002 (20 μM) and wortmannin (100 nM) to study if suppression of PI3K/Akt activity could reverse its resistance to taxol-induced apoptosis. As shown in Figure 2b, after treatment with LY294002, the level of taxol-induced p-Akt was decreased in the cells treated with both taxol and LY294002 (Lanes 4, 7) compared to the cells treated with taxol alone (Lanes 3, 6) at both time points examined. This inhibitory effect of LY294002 was associated with an appearance of cleaved-PARP expression at 24 hr post-exposure time (Lane 7 vs. Lane 6). Similar results were observed in the cells treated with wortmannin (Fig. 2c, Lane 3 vs. Lane 2). These results demonstrate that suppression of taxol-induced PI3K/Akt activity led to increased sensitivity to taxol-induced apoptosis in HNE1-T3 cells. To confirm the positive role of p38MAPK in taxol-induced apoptosis, we then treated HNE1 cells with a p38MAPK inhibitor, SB203580 (20 μM). As shown in Figure 2d, the SB203580 treatment suppressed the taxol-induced p38MAPK phosphorylation (Lanes 4, 7 vs. Lanes 3, 6) which was correlated with disappearance of cleaved-PARP (Lane 7 vs. Lane 6) after exposure to taxol for 24 hr, suggesting that inhibition of p38MAPK activity is able to confer resistance to taxol-induced apoptosis in HNE1 cells. An inactive analog of the p38MAPK inhibitor, SB202474 (20 μM) was also used to treat HNE1 cells as a negative control. As shown in Figure 2e, SB202474 treatment did not inhibit p38MAPK phosphorylation which was associated with increased expression of cleaved-PARP. These results implicate that Akt and p38MAPK pathways may play a key role in the TWIST-mediated taxol resistance in these cells.
Effect of taxol on cell cycle distribution in HNE1 and HNE1-T3 cells
It is generally believed that the antitumour effect of taxol is mainly through its interference with microtubules which leads to mitotic arrest and subsequent cell death.25 To investigated if upregulation of TWIST played a role in taxol-induced cell cycle alterations, we performed flow cytomeric analysis on HNE1 and HEN1-T3 cells. First, we treated these two cell lines with their corresponding IC50 (Inhibition Concentration of 50% cell survival; IC50HNE1 = 0.2 ng/ml; IC50HNE1-T3 = 0.45 ng/ml) and IC90 (Inhibition Concentration of 90% cell survival; IC90HNE1 = 2.6 ng/ml; IC90HNE1-T3 = 6.0 ng/ml) concentrations to study if same cytotoxic doses of taxol could lead to differential cell cycle distribution between these 2 cell lines. As shown in Figure 3a, no G2/M arrest was observed in either cell lines treated with 2 concentrations of taxol at 3 time points (24, 48 and 72 hr), indicating that upregulation of TWIST did not play a role in cell cycle distribution in response to taxol. In fact, there was a decrease in the percentage of G2/M phase cells in a time dependent manner in both cell lines which was associated with an increased G1 phase fraction. To further confirm these results, we then treated these 2 cell lines with 2 relatively high doses of taxol (5 ng/ml; 10 ng/ml) and then studied cell cycle distribution. As shown in Figure 3b, as expected, the sensitive HNE1 cells showed a high percentage of sub-G1 phase cells, indicating large number of cells undergoing apoptosis in a time and dose-dependent manner. On the contrary, the percentage of sub-G1 phase cells was much lower in HNE1-T3 cells in response to the same doses. However, with an exception of a transient G2/M arrest in both cell lines treated with relatively high dose (10 ng/ml, 12 hr post-exposure time), we did not observe any close association between G2/M arrest and sensitivity to taxol in these 2 cell lines. These results suggest that the TWIST-induced taxol resistance in HNE1-T3 cells is not associated with taxol-induced G2/M arrest.
Correlation of TWIST expression and cellular sensitivity to taxol and vincristine in NPC cell lines
To further confirm the significance of TWIST in cellular sensitivity to microtubule disrupting agents, we then examined TWIST expression and cellular sensitivity to taxol and vincristine in additional 8 NPC cell lines, CNE1, CNE2, CNE3, HONE1, TW01, TW04, SUNE1 and HK1. As shown in Figure 4a, 7 out of 8 cell lines (87.5%) expressed TWIST at different levels, with the highest levels in CNE2, HONE1 and HK1 cell lines. Colony forming assay presented in Figure 4b showed that the 3 cell lines with high TWIST expression (solid lines) were more resistant to both anticancer drugs, especially to taxol. In contrast, the cell lines with relatively lower TWIST (dotted lines) were much more sensitive to both drugs, especially CNE3 which showed undetectable levels of TWIST and most sensitive to both drugs. These results suggest that upregulation of TWIST is associated with resistance to microtubule disrupting agents, especially to taxol in NPC cell lines.
Effect of TWIST inactivation on taxol sensitivity in NPC cells
Microtubule disrupting anticancer drugs, especially taxol, are widely used in the treatment of human cancer and development of resistance is a frequent clinical problem. To explore the possibility of using TWIST as a target to increase taxol-induced cancer cell death, we inactivated TWIST in 3 NPC cell lines (CNE1, CNE2, HONE1) through small RNA interference and studied if suppression of TWIST could lead to increased sensitivity to taxol-induced apoptosis. As shown in Figures 5a and 5b, transfection of Si-TWIST (Si-T) in the 3 NPC cell lines led to suppression of TWIST expression at both mRNA (Fig. 5a) and protein (Fig. 5b) levels compared to the controls (Si-C). MTT assay showed that the decreased TWIST expression in the transfectants was associated with decreased cell viability in a dose dependent manner after exposure to 5 concentrations of taxol for 24 hr (Fig. 5c, open columns). Western blotting analysis showed that inactivation of TWIST in the Si-TWIST transfectants led to suppression of p-Akt expression in all 3 cell lines compared to the control (Fig. 5d, time 0 hr). After exposure to taxol (10 ng/ml), the differential p-Akt expression became more prominent which was associated with increased cleaved-PARP in the Si-TWIST transfectants (Fig. 5d). However, we did not observe any significant association in p38MAPK levels between the cells with high or low levels of TWIST (data not shown). These results further confirm the importance of Akt pathway in the TWIST-mediated taxol resistance and suggest that inactivation of TWIST may be a potential therapeutic target in reversing taxol resistance in NPC cells.
In this study, we demonstrated that TWIST acted as an anti-apoptotic factor in inducing taxol resistance in NPC cells. The findings that activation of Akt kinase was associated with TWIST-mediated taxol resistance suggest that increased TWIST in NPC cells may be able to activate the Akt pathway which in turn suppresses taxol-induced apoptosis leading to drug resistance. Since upregulation of TWIST has been correlated with advanced stage, poor survival as well chemodrug resistance in cancer patients,9, 14, 15, 16, 17 our findings provide a possible underlying mechanism responsible for its action and a therapeutic target for overcoming taxol resistance in cancer cells.
Recently, TWIST has been reported to participate in the regulation of cell survival. For example, TWIST-null mice show a massive wave of apoptosis in the sites where TWIST is normally expressed.2 In addition, mutations causing TWIST haploinsufficiency in human calvarial osteoblasts lead to promotion of apoptosis.5 On the other hand, ectopic TWIST expression is able to protect mouse fibroblast cells from apoptosis.7 Therefore, it has been suggested that the anti-apoptotic role of TWIST in human calvarial osteoblasts, together with alterations in osteoblast proliferation and differentiation, may play a key role in the development of Saethre-Chotzen and mutational inactivation of TWIST may result in osteoblast apoptosis leading to premature suture ossification.6 Although several signalling pathways such as ARF/MDM2/p53, TNFα and IGF have been reported to be associated with its anti-apoptotic effect in osteoblasts and fibroblasts,5, 7, 26 molecular mechanisms responsible for its role in the development and progression of human cancer, especially chemodrug resistance, are largely unknown. In this study, we demonstrated that high TWIST expression in the resistant HNE1-T3 cells was not only correlated with suppression of taxol-induced apoptosis (Fig. 1) but also associated with increased p-Akt and suppression of p-p38MAPK expression (Fig. 2a). In addition, the fact that treatment with PI3K/Akt inhibitors, LY294002 or wortmannin, was able to promote taxol-induced apoptosis in HNE1-T3 cells evidenced by the appearance of cleaved-PARP (Figs. 2b and 2c), suggests that Akt activation may play a key role in the TWIST-mediated taxol resistance. This hypothesis was further supported by the small RNA interference studies on additional NPC cell lines that downregulation of TWIST was associated with suppression of Akt phosphorylation and increased sensitivity to taxol-induced apoptosis (Fig. 5). Although results on HNE1-T3 cells showed a significance of p38MAPK suppression in the TWIST-mediated taxol resistance (Figs. 2a and 2c), we did not observe any close association between p38MAPK and cellular sensitivity in the Si-TWIST transfectants (data not shown). It is possible that suppression of p38MAPK observed in HNE1-T3 cells may be a specific event in this cell line or a consequence of long term drug selection during generation of acquired resistance. Nevertheless, our results indicate that the TWIST-mediated drug resistance may be regulated through its positive interaction with the Akt pathway leading to suppression of taxol-induced apoptosis in NPC cells. Although the precise molecular mechanism responsible for the TWIST-induced Akt activation is not clear, recently, a possible link between TWIST and Akt pathway has been suggested. For example, Akt has been shown to interferer with p53/MDM2 pathway,27, 28 which is also a target of TWIST.7 In addition, it was reported that inactivation of STAT3 in a breast cancer cell line, 4T1, led to downregulation of Akt-phosphorylation which was associated with completely abolished TWIST protein expression.29 Furthermore, a separate study reported that another effector of STAT3, c-Myc, was able to co-operate with TWIST to override p53-dependent apoptosis leading to malignant transformation of primary MEF.30 Since both STAT3 and Akt pathways are crucial in controlling cell survival and protection against apoptosis in cancer cells,31 it is highly possible that the anti-apoptotic function of TWIST may be mediated through interaction with these pathways. Currently, we are in the process of confirming this hypothesis.
Disruption of mitosis through interfering with microtubules is one of the main targets of taxol-induced cytotoxicity in cancer cells. It is believed that the taxol-induced mitotic arrest is a trigger for taxol-induced cell death.25 In addition, recently, upregulation of TWIST has been reported to be associated with chromosomal instability in breast cancer specimens.12 However, in the present study, in spite of treating HNE1 and HNE1-T3 cell lines with different doses but same cytotoxic effect of taxol (IC50 or IC90 concentrations), and relatively high doses (5, 10 ng/ml), we did not observe any association between G2/M phase accumulation and taxol sensitivity in both cell lines (Fig. 3). This may be due to the fact that the mitotic checkpoint control is partially defective in HNE1 cells as a results of its relatively low expression of a key regulator, MAD2.21 These results further support our previous suggestion that the TWIST-mediated taxol resistance is not regulated through its interference with mitosis but its role as an anti-apoptotic factor.19
Microtubule disrupting anticancer drugs have been used widely in the treatment of many types of human cancer. However, with initial high response rates, resistance often develops in the majority of patients with advanced disease. Therefore, early identification of potentially resistant patients may be able to facilitate clinical management of cancer patients. In this study, our findings that high levels of TWIST expression were associated with resistance to taxol and vincristine in NPC cell lines (Fig. 4) suggest a potential marker for identification of patients with poor clinical response. This hypothesis is also supported by a recent study which reported that upregulation of TWIST in osteosarcoma patients was associated with poor response to chemotherapeutic drugs, and suggested that TWIST may be used as a predictor for poor clinical response.17 Since overcoming drug resistance is a main obstacle in the successful treatment of human cancer, our results that downregulation of TWIST led to increased sensitivity to taxol-induced apoptosis and cell viability (Fig. 5) provide a molecular target in reversing taxol resistance in cancer cells. In addition, as the PI3K/Akt signally pathway has been considered as an attractive target for the development of novel anticancer drugs, our results also implicate TWIST as an alternative target for the Akt signaling pathway.
In summary, the current study demonstrates an underlying molecular mechanism responsible for the TWIST-mediated chemodrug resistance as well as its role in cancer progression. Our findings also suggest a potential marker for identification of tumours that are likely to be resistant to microtubule disrupting agents especially taxol and provide a target for overcoming taxol resistance in cancer cells.
This work was supported by grants (Association for International Cancer Research, UK) to Ms. X. Wang.