Advanced non–small cell lung cancer (NSCLC) has a dismal prognosis. βIII-Tubulin, a protein highly expressed in neuronal cells, is strongly associated with drug-refractory and aggressive NSCLC. To date, the role of this protein in in vivo drug resistance and tumorigenesis has not been determined. NSCLC cells stably expressing βIII-tubulin short hairpin RNA displayed reduced growth and increased chemotherapy sensitivity when compared with control clones. In concordance with these results, stable suppression of βIII-tubulin reduced the incidence and significantly delayed the growth of tumors in mice relative to controls. Our findings indicate that βIII-tubulin mediates not only drug sensitivity but also the incidence and progression of lung cancer. βIII-Tubulin is a cellular survival factor that, when suppressed, sensitizes cells to chemotherapy via enhanced apoptosis induction and decreased tumorigenesis. Findings establish that upregulation of a neuronal tubulin isotype is a key contributor to tumor progression and drug sensitivity in lung adenocarcinoma. Cancer Res; 70(12); 4995–5003. ©2010 AACR.
Advanced non–small cell lung cancer (NSCLC) has a dismal prognosis and remains the most common cause of cancer-related death worldwide. Mechanisms mediating resistance and tumor aggressiveness are poorly defined. βIII-Tubulin, a microtubule protein highly expressed in neuronal cells, is strongly associated with drug-refractory and aggressive NSCLC (1). Microtubules are multifunctional cytoskeletal proteins involved in many essential cellular roles, including maintenance of cell shape, intracellular transport, and in mitosis, forming mitotic spindles to ensure proper chromosome segregation and cell division. The soluble α/β-tubulin heterodimers assemble to form the microtubule polymer, with the soluble and polymer forms coexisting in a state of dynamic equilibrium (2). A number of α- and β-tubulin isotypes have been identified that display differential developmental and tissue expression and differ in their chromosomal localization (2). It is increasingly apparent that the cellular role of microtubules extends beyond structural support into key signaling and apoptotic roles.
β-Tubulin is the cellular target of clinically important tubulin-binding agents (TBA) used in cancer therapy. High expression of βIII-tubulin was associated with resistance in paclitaxel-selected NSCLC cells and clinical resistance to paclitaxel in ovarian cancer (3). Translational studies have now clearly established that expression of βIII-tubulin is associated with resistance to taxanes or vinorelbine in a range of tumor types, including lung, ovarian, breast, gastric, and cancers of unknown origin (reviewed in refs. 1, 4). To date, resistance mediated by βIII-tubulin was thought to be restricted to TBAs. However, we recently identified a broader role for βIII-tubulin in NSCLC (5). βIII-Tubulin can mediate response in vitro not only to broad classes of TBAs but also to DNA-damaging agents. This broad chemosensitization is specific to βIII-tubulin, as silencing of βII- or βIVb-tubulin does not sensitize NSCLC cells to paclitaxel (6). In addition, βIII-tubulin under stress conditions has been found to be a mediator of cell survival (7). Indeed, it has been known for some time that tumors expressing βIII-tubulin have a poorer clinical outcome than tumors that have low or no expression of this tubulin isotype (reviewed in refs. 1, 8). In lung and a number of other cancers, increased expression of βIII-tubulin is associated with poorly differentiated tumor tissue, high-grade malignancy, and metastatic potential (8, 9). A recent study found that completely resectable NSCLC tumors, with high expression of βIII-tubulin, correlated with resistance to docetaxel. Collectively, the laboratory and clinical data strongly suggest that βIII-tubulin may have a broader role in the tumorigenesis of certain cancers, such as NSCLC (10). Major issues that have yet to be resolved include the role of βIII-tubulin in mediating in vivo drug sensitivity and whether βIII-tubulin is functionally involved in the tumorigenic phenotype of epithelial cancers.
Herein, we show that βIII-tubulin is mediating in vitro and in vivo drug sensitivity. Importantly, suppression of βIII-tubulin expression reduces anchorage-independent growth and leads to decreased incidence and progression of NSCLC cell xenografts, directly implicating this protein in the tumorigenic phenotype of NSCLC.
Materials and Methods
Generation of βIII-tubulin stable short hairpin RNA–expressing cells
The human NSCLC cell line H460 was maintained as previously described (5). For the generation of βIII-tubulin stable knockdown cells, H460 cells were transfected with the pRS vector containing the βIII-tubulin short hairpin RNA (shRNA) expression cassette (pRS/βIIISH) and the pRS vector containing a noneffective shRNA cassette against green fluorescent protein that acts as a negative control (pRS/CtrlSH; OriGene Technologies, Inc.). The 29-mer shRNA sequence that targets βIII-tubulin is as follows: 5′-GTGTGAGCTGCTCCTGTCTCTGTCTTATT-3′. Cells were selected in growth media containing puromycin (Sigma-Aldrich) to enrich the population of cells that had retained the expression plasmid. Approximately 6 to 60 individual clonal populations were isolated for each construct. After clonal expansion, each clone was examined for βIII-tubulin expression by Western blotting as described below.
Gene expression of βIII-tubulin by real-time PCR
The expression of βIII-tubulin in stably expressing shRNA cells was examined using real-time quantitative PCR. Total RNA was extracted and DNase treated using the Qiagen RNeasy Plus kit according to the manufacturer's instructions. Real-time PCR was done using the QuantiTect SYBR Green PCR kit (Qiagen). βIII-Tubulin mRNA sequences used were as follows: forward, 5′-GCGAGATGTACGAAGACGAC-3′; reverse, 5′-TTTAGACACTGCTGGCTTCG-3′. All data were normalized to the housekeeping gene β2-microglobulin (β2-Microglobulin QuantiTect Primer Assay, Qiagen).
Western blotting of tubulin isotypes
Western blot analyses using the following antibodies were performed as described previously (5): βIII-tubulin (clone TUJ1; Chemicon), βI-tubulin (clone SAP 4G5; Abcam Ltd.), βII-tubulin (clone 7B9; Chemicon), βIV-tubulin (clone ONS. 1A6; Sigma), total tubulin (clone TUB 2.1; Abcam), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; clone 6C5; Abcam).
Assessment of drug sensitivity in vitro
Drug-treated clonogenic assays using CDDP or paclitaxel were performed as described previously (5).
Assessment of drug sensitivity in vivo
BALB/c nude mice (6–8 wk old) were obtained from the Animal Resource Center at the University of New South Wales (Sydney, New South Wales, Australia) and maintained under specific pathogen-free conditions for the studies. All animal experiments were approved by the Animal Ethics Committee, University of New South Wales (ACEC#: 07/89B). Stably expressing βIII-tubulin shRNA (pRS/βIIISH4 or pRS/βIIISH59) or control (pRS/CtrlSH1 and pRS/CtrlSH2) cells (1 × 106) were inoculated s.c. Once tumors reached approximately 200 to 300 mm3, mice were randomized into treatment groups (five mice per treatment group) and treated with 3.33 mg/kg CDDP (one injection once a day for 3 d) or its vehicle control (PBS). Mice were then closely monitored and tumors were measured twice weekly. Tumor volume was calculated using the formula (a × b × c)/2, where a and b are the shorter and longer diameter, respectively, of the tumors and c is the depth. All mice were sacrificed once tumors reached 1,000 mm3 or if they lost ≥20% body weight. At the time of sacrifice, tumor tissue was collected and placed into 4% paraformaldehyde for histologic analysis or snap frozen in liquid N2 for biochemical analysis.
Rescue of βIII-tubulin expression in shRNA-expressing NSCLC cells
Full-length Hβ4 was amplified from a L1110, pMOSBlue vector construct (Amersham Biosciences) containing Hβ4 cDNA, with the following primers: forward, 5′-TAATACGACTCACTATAGGG; reverse, 5′-ATTCATTCGCGGCCGCCACC. PCR amplification was performed using the FailSafe PCR System (Epicentre Biotechnologies). The amplified product was subcloned into the episomal expression vector, pREP4 (Invitrogen), following digestion with HindIII and NotI restriction enzymes (Promega). This resulted in the introduction of an additional methionine start site that was subsequently removed by amplifying the construct with the following primers: forward, 5′-TCATAAGCTTATGCGGGAGATCGTGC; reverse, 5′-TGCCTCTCGCGGCCGCTGCGAGCAGC. The amplified product was then subcloned into pREP4 again following digestion with HindIII and NotI to generate the plasmid pREP4/βIII. For isolation of stable rescue clones, two independent stable βIII-tubulin shRNA clones (pRS/βIIISH4 or pRS/βIIISH59) were transfected with the pREP4/βIII vector (pRS/βIIISH4/pREPR6, pRS/βIIISH4/pREPR17, pRS/βIIISH59/pREPR33, and pRS/βIIISH59/pREPR37) or the empty pREP4 vector (pRS/βIIISH4/pREPEV1 and pRS/βIIISH4/pREPEV2) as a control. Cells were then selected in media containing hygromycin B (Calbiochem). Individual colonies were isolated and expanded. The resultant cells were assessed for βIII-tubulin by Western blotting as described above.
Immunohistochemistry was carried out to measure the expression of βIII-tubulin and Ki67 in paraffin-embedded subcutaneous tumor tissue using a βIII-tubulin monoclonal rabbit antibody (clone TUJ1 1-15-79; Covance) and a mouse monoclonal Ki67 antibody (clone MIB-1; Dako). 3,3′-Diaminobenzidine was used as a substrate for the peroxidase reaction and hematoxylin as the counterstain. The specificity of the primary antibodies was confirmed by including several negative controls: (a) omission of the primary antibody and (b) incubating sections with normal goat IgG (Vector Laboratories) at the same concentration as the primary antibody.
In vitro tumorigenesis assay
Cells stably expressing βIII-tubulin (pRS/βIIISH60, pRS/βIIISH59, and pRS/βIIISH4) or control (pRS/CtrlSH1 and pRS/CtrlSH2) shRNA were resuspended in 0.33% agar in growth media and plated on 0.5% solidified agar (bottom supportive layer). Triplicate plates were set up for each sample. After 12 days of culture, individual colonies were counted and photographed using the Zeiss Axiovert S100 inverted microscope and SPOT digital camera. The results were expressed as the percentage of colony formation, according to the formula: (number of colonies formed/number of cells seeded) × 100%.
In vivo tumorigenesis assay
BALB/c nude mice (6–8 wk old) were inoculated s.c. with cells stably expressing βIII-tubulin (pRS/βIIISH4 or pRS/βIIISH59) or control (pRS/CtrlSH1 and pRS/CtrlSH2) shRNA (1 × 106) cells (10 mice per group). Tumors were measured twice weekly using digital calipers, and tumor volume was calculated using the formula (a × b × c)/2, where a and b are the shorter and longer diameter, respectively, of the tumors and c is the depth. All mice were sacrificed once tumors reached 1,000 mm3 or if mice had lost ≥20% body weight. At the time of sacrifice, tumors were divided into sections and placed into 4% paraformaldehyde for histologic analysis or snap frozen in liquid N2 for biochemical analysis.
Data are expressed as the mean ± SE and analyzed using ANOVA or Student's t test followed by the nonparametric Dunnett or Mann-Whitney tests using the GraphPad Prism program. Survival curves were plotted by the Kaplan-Meier method and tested for differences with the log-rank statistic. A P value of <0.05 was considered statistically significant.
Generation of stable βIII-tubulin shRNA-expressing NSCLC cells
To allow a long-term study of the possible effects of silencing βIII-tubulin expression on in vivo drug sensitivity and the tumorigenic potential, NSCLC cells were generated, which stably express the pRS/βIIISH construct (refer to Materials and Methods). Control cells were transfected with a nonfunctional 29-mer shRNA. Three independent βIII-tubulin–expressing shRNA clones, designated pRS/βIIISH60, pRS/βIIISH59, and pRS/βIIISH4, were identified as cells that stably express significantly reduced levels (>80% knockdown) of βIII-tubulin at both the gene transcript and protein level when compared with the control clones pRS/CtrlSH1 and pRS/CtrlSH2 (Supplementary Fig. S1A and B). Specific stable knockdown of βIII-tubulin was achieved without compensatory upregulation of other β-tubulin isotypes (Supplementary Fig. S2). This is not surprising given that βIII-tubulin makes up ∼7% of the total β-tubulin pool in H460 cells (11).
Inhibition of βIII-tubulin increases sensitivity to cisplatin in NSCLC cells both in vitro and in vivo
Previously, we have shown that transient knockdown of βIII-tubulin using conventional small interfering RNA (siRNA) significantly sensitized NSCLC cells to both TBAs and DNA-damaging agents such as cisplatin (CDDP; ref. 5). To confirm that stable βIII-tubulin shRNA-expressing NSCLC cells behaved in a similar fashion, we assessed their sensitivity to CDDP (a platinum-based agent that is often used as the backbone for systemic chemotherapy in the treatment of NSCLC). All three βIII-tubulin knockdown clones (pRS/βIIISH60, pRS/βIIISH59, and pRS/βIIISH4) were found to exhibit increased sensitivity to CDDP (Fig. 1A). pRS/βIIISH4, which has the greatest amount of knockdown, showed the greatest sensitivity to CDDP (mean ID50 value, 0.17 μmol/L). Similarly, pRS/βIIISH60 and pRS/βIIISH59 were significantly more sensitive to CDDP (mean ID50 values of 0.22 and 0.24 μmol/L, respectively) compared with control clones (pRS/CtrlSH1 and pRS/CtrlSH2; mean ID50 values of 0.41 and 0.38 μmol/L, respectively). Similar results were observed when cells were treated with the TBA paclitaxel or the cisplatin analogue carboplatin (Supplementary Fig. S3A and B).
Rescue of βIII-tubulin abolishes increased sensitivity to cisplatin
To verify the specificity of reduced levels of βIII-tubulin on chemosensitivity and to rule out any potential off-target effects of shRNA, we performed rescue experiments. Briefly, βIII-tubulin shRNA-expressing (pRS/βIIISH4 or pRS/βIIISH59) cells were transfected with the full-length human cDNA of the βIII-tubulin gene with expression driven by the episomal vector pREP4. Cells were also transfected with the empty vector alone as a control. Four independent βIII-tubulin rescue clones were chosen from the βIII shRNA stably expressing clones pRS/βIIISH4 and pRS/βIIISH59 and designated as pRS/βIIISH4/pREPR6, pRS/βIIISH4/pREPR17, pRS/βIIISH59/pREPR33, and pRS/βIIISH59/pREPR37. All four rescue clones showed restored βIII-tubulin protein to levels similar to controls (pRS/CtrlSH1 and pRS/CtrlSH2; Fig. 1B). No effect on βIII-tubulin levels was observed for cells stably expressing the empty pREP4 vector (pRS/βIIISH4/pREPEV1 and pRS/βIIISH4/pREPEV2; Fig. 1B). Importantly, sensitivity to CDDP was completely restored in all four βIII-tubulin rescue clones (pRS/βIIISH4/pREPR6, mean ID50 = 0.59 μmol/L; pRS/βIIISH4/pREPR17, mean ID50 = 0.41 μmol/L; pRS/βIIISH59/pREPR33, mean ID50 = 0.41 μmol/L; pRS/βIIISH59/pREPR37, mean ID50 = 0.42 μmol/L) when compared with cells expressing the empty pREP4 vector (pRS/βIIISH4/pREPEV1, mean ID50 = 0.20 μmol/L; pRS/βIIISH4/pREPEV2, mean ID50 = 0.16 μmol/L; pRS/βIIISH59/pREPEV1, mean ID50 = 0.14 μmol/L; pRS/βIIISH59/pREPEV2, mean ID50 = 0.13 μmol/L). In fact, the ID50 values for the rescue clones were very similar to the control shRNA cells (pRS/CtrlSH1, mean ID50 = 0.42 μmol/L; pRS/CtrlSH2, mean ID50 = 0.34 μmol/L in Fig. 1A; also refer to Fig. 1C and D). Collectively, these results confirm that reduced levels of βIII-tubulin are responsible for the increased sensitivity to CDDP in NSCLC.
Inhibition of βIII-tubulin increases sensitivity to apoptosis in the presence of cisplatin
To understand the basis for the increased drug sensitivity following suppression of βIII-tubulin expression, cell death pathways were investigated. The increase in sensitivity to CDDP was associated with an enhanced induction of apoptosis in the βIII-tubulin shRNA-expressing cells as evidenced by a significant increase in Annexin V (a measure of phosphatidylserine externalization) staining (Supplementary Fig. S4A). In addition, to explore whether the intrinsic or extrinsic apoptotic pathways were involved, we measured the activity of the initiator caspase-8 and caspase-9 after exposure to CDDP. βIII-Tubulin shRNA-expressing cells and their controls displayed an increase in caspase-9 activity when exposed to 2 and 4 μmol/L CDDP (Supplementary Fig. S4B). However, there was no significant difference in activity between the βIII-tubulin shRNA-expressing and control shRNA-expressing cells (Supplementary Fig. S4B). In contrast, a significant increase in caspase-8 activity was observed in the βIII-tubulin shRNA-expressing cells when compared with controls after exposure to CDDP (Supplementary Fig. S4C). This increase in activity also correlated with a significant increase in the activation of the effector caspase-3/caspase-7 and cleavage of its substrate poly(ADP-ribose) polymerase (PARP) in the βIII-tubulin shRNA-expressing cells (Supplementary Fig. S5). Collectively, these data suggest that inhibiting βIII-tubulin enhances drug-induced apoptosis via a caspase-mediated cascade in lung cancer cells.
Inhibition of βIII-tubulin increases sensitivity to cisplatin in vivo
Having shown that βIII-tubulin suppression increased in vitro drug sensitivity, we sought to determine whether reduced levels of βIII-tubulin could sensitize NSCLC tumors to CDDP in an in vivo setting. BALB/c nude mice were xenografted with βIII-tubulin (pRS/βIIISH4 or pRS/βIIISH59) or control (pRS/CtrlSH1 and pRS/CtrlSH2) shRNA-expressing NSCLC cells. Once tumors reached approximately 200 to 300 mm3, mice were treated with CDDP or its vehicle. Mice injected with βIII-tubulin shRNA-expressing cells (pRS/βIIISH4 or pRS/βIIISH59) displayed a significant delay in tumor growth (time to reach 1,000 mm3) and increased median survival after treatment with CDDP when compared with control shRNA-expressing (pRS/CtrlSH1 and pRS/CtrlSH2) tumors [pRS/CtrlSH1 (vehicle, PBS) = 15.6 d; pRS/CtrlSH1 (CDDP) = 13 d; pRS/CtrlSH2 (vehicle, PBS) = 7.2 d; pRS/CtrlSH2 (CDDP) = 10.8 d; pRS/βIIISH4 (vehicle, PBS) = 15.6 d; pRS/βIIISH4 (CDDP) = 35 d; pRS/βIIISH59 (vehicle, PBS) = 15.6 d; pRS/βIIISH59 (CDDP) = 29.4 d; Fig. 2A–C]. Taken together, these results show that the level of βIII-tubulin regulates drug sensitivity in NSCLC and that suppressing βIII-tubulin expression confers a survival advantage after chemotherapy treatment in vivo.
Knockdown of βIII-tubulin decreases anchorage-independent growth in NSCLC cells
The ability of transformed cells to grow under anchorage-independent conditions is one of the hallmark properties that are associated with the tumorigenic potential of a cancer cell (12). Given that increased levels of βIII-tubulin are associated with more aggressive disease in NSCLC, we then proceeded to assess whether βIII-tubulin plays a role in tumorigenicity. βIII-Tubulin knockdown clones (pRS/βIIISH4, pRS/βIIISH59, and pRS/βIIISH60) were grown in soft agar, and the number of colonies formed was assessed. Stable knockdown of βIII-tubulin significantly reduced the number of colonies formed in all three βIII-tubulin knockdown clones (pRS/βIIISH4, mean % colony formation = 15.89; pRS/βIIISH59, mean % colony formation = 33.39; pRS/βIIISH60, mean % colony formation = 32.88) when compared with control (pRS/CtrlSH1, mean % colony formation = 58.12; pRS/CtrlSH2, mean colony % formation = 59.82; Fig. 3A). Similar results were also observed in cells treated with two different 27-mer dicer siRNA targeting different regions of the βIII-tubulin gene as well as conventional siRNA targeting βIII-tubulin (Supplementary Fig. S6A). Notably, the effect of reduced βIII-tubulin on tumorigenicity seemed to be tubulin isotype specific, as no significant effect was observed when cells were treated with siRNA targeting another β-tubulin isotype, βIVb-tubulin (Supplementary Fig. S6B). This suggests that βIII-tubulin may play a novel and specific role in the tumorigenic potential of NSCLC cells.
Rescue of βIII-tubulin expression restores anchorage-independent growth of NSCLC cells
To verify the specificity of βIII-tubulin in contributing to tumorigenicity, the βIII-tubulin in the stable knockdown clones pRS/βIIISH59 and pRS/βIIISH4 was rescued as described above. Restoration of βIII-tubulin effectively rescued the suppression of anchorage-independent growth in the NSCLC cells (pRS/βIIISH4/pREPR6, mean % colony formation = 48.05; pRS/βIIISH4/pREPR17, mean % colony formation = 45.50; difference = 35.12; Fig. 3B). In contrast, empty pREP vector clones that maintained reduced βIII-tubulin expression (pRS/βIIISH4/pREPEV1 and pRS/βIIISH4/pREPEV2) failed to restore the clonogenic growth in soft agar (pRS/βIIISH4/pREPEV1, mean % colony formation = 10.38%; pRS/βIIISH4/pREPEV2, mean % colony formation = 10.33%) compared with control shRNA clones (pRS/CtrlSH1 and pRS/CtrlSH2; Fig. 3A). These results provide strong support that the decrease in anchorage-independent growth is directly attributable to the reduction of βIII-tubulin expression.
Suppression of βIII-tubulin decreases the incidence and progression of tumor growth in vivo
To examine whether reduced levels of βIII-tubulin could influence tumor incidence and growth in vivo, we injected mice with NSCLC cells stably expressing the βIII-tubulin shRNA construct (pRS/βIIISH59 and pRS/βIIISH4) or its control (pRS/CtrlSH1 and pRS/CtrlSH2). Tumor development and growth was recorded over a 12-week period. Mice injected with control shRNA (pRS/CtrlSH1 and pRS/CtrlSH2) cells had a 100% (20 of 20) tumor incidence. However, only 65% (13 of 20) of mice injected with βIII-tubulin shRNA-expressing cells (pRS/βIIISH59 and pRS/βIIISH4) formed tumors after 12 weeks (Fig. 4A). To confirm that βIII-tubulin shRNA was active, βIII-tubulin expression was assessed in tumor tissue. Importantly, βIII-tubulin mRNA and protein levels were significantly reduced in βIII-tubulin shRNA-expressing tumors (pRS/βIIISH59 and pRS/βIIISH4) when compared with control (pRS/CtrlSH1 and pRS/CtrlSH2; Fig. 4B and C). The decrease in βIII-tubulin expression in tumor tissue was also confirmed by immunohistochemistry (Fig. 4D and E). Moreover, tumors expressing βIII-tubulin shRNA showed a significant delay in tumor growth (βIII-tubulin shRNA, 67.8 d versus control shRNA, 36.15 d) and size when compared with control tumors (Fig. 5A and B). Taken together, these results confirm that the activity of βIII-tubulin shRNA is highly potent and active in an in vivo setting. More importantly, these results clearly show for the first time that βIII-tubulin has an important functional role in tumor development and progression in NSCLC.
βIII-tubulin is predominantly a neuronal-expressed cytoskeletal protein that is associated with drug resistance and aggressive tumors in a range of cancer types, including NSCLC, ovarian, and breast cancers (1, 4). Despite strong correlative preclinical and clinical evidence implicating a role for βIII-tubulin in tumorigenesis, its role in tumor formation and aggression has not been addressed (1, 13, 14). Herein, we have identified and validated a new mechanism of action for βIII-tubulin as a cellular survival factor that, when suppressed, sensitizes cells to chemotherapy via enhanced apoptosis induction and decreased tumorigenesis.
Previous studies have attempted to address the role of βIII-tubulin in drug resistance by overexpressing βIII-tubulin (15, 16). However, the results have been difficult to interpret due to increased cell toxicity and significant compensatory changes in the levels of other β-tubulin isotypes. In this study, we overcame a number of these challenges by using a RNA interference approach and examined the effects of stable knockdown of βIII-tubulin on drug sensitivity. Consistent with transient knockdown of βIII-tubulin in two independent NSCLC cell lines (5), stable knockdown of βIII-tubulin resulted in increased in vitro sensitivity to cisplatin, carboplatin, and paclitaxel. Enhanced sensitivity to cisplatin in the βIII-tubulin knockdown cells was correlated with a significant induction of drug-induced apoptosis, as evidenced by an increase in Annexin V staining as well as increased activity of the initiator caspase-8 and the downstream effector caspase-3/caspase-7 and its substrate PARP, suggesting that suppressing βIII-tubulin levels in NSCLC may increase cell death on exposure to chemotherapy by modulating caspase activity. Importantly, confirmation that suppression of βIII-tubulin was directly responsible for the enhanced sensitivity to CDDP and not off-target effects of shRNA was obtained when βIII-tubulin levels were “rescued” back into the cells.
Given the strong clinical evidence linking high βIII-tubulin levels and drug resistance, our finding that stable suppression of βIII-tubulin expression increases in vivo sensitivity to CDDP has direct clinical relevance. Taken together with the in vitro data showing increased susceptibility to drug-induced apoptosis in βIII-tubulin knockdown cells, βIII-tubulin seems to be mechanistically involved as a survival factor, which helps protect cancer cells from cell death by chemotherapy drugs. In support, a recent study in ovarian cancer cells exposed to the stress condition hypoxia showed that βIII-tubulin expression was significantly increased (17) and hypoxia-induced βIII-tubulin expression correlated with paclitaxel resistance (18). In our model, we cannot exclude the possibility that βIII-tubulin may be induced under hypoxic conditions, as the potent stable knockdown would mask any effects. Of note is that a number of signaling proteins involved in regulating drug resistance coimmunoprecipitate or colocalize with βIII-tubulin (17, 19). Therefore, it is feasible that silencing βIII-tubulin expression in cancer cells disrupts important protein interactions and signaling processes, which are vital for providing cancer cells with a survival advantage when exposed to cytotoxic stressors.
High levels of βIII-tubulin in clinical samples are correlated strongly with a poorly differentiated and invasive tumor phenotype in a number of epithelial-derived cancers, including NSCLC. βIII-Tubulin expression was positively correlated with decreased overall survival in ovarian and NSCLC patients regardless of response to chemotherapy, suggesting that βIII-tubulin may contribute to the aggressive behavior of a tumor rather than only a marker of chemotherapy resistance (1, 20). However, despite strong correlative evidence implicating βIII-tubulin in tumorigenesis, its role in tumor incidence and development has been lacking. This study provides the first evidence that βIII-tubulin levels directly influence anchorage-independent growth (a measure of tumorigenic potential). This raises an important question: Is this phenotype specific to suppression of βIII-tubulin or would another β-tubulin isotype mediate a similar effect? The phenotype seems to be specific to βIII-tubulin, as no effect was observed in cells treated with siRNA targeting another β-tubulin isotype, βIVb-tubulin (Supplementary Fig. S6B). However, it cannot be ruled out that other β-tubulin isotypes may also affect tumorigenicity. We also addressed the possibility that the βIII-tubulin shRNA was mediating “off-target” effects. However, rescue of βIII-tubulin restored anchorage-independent growth to control levels, again confirming that the observed effect was a direct result of βIII-tubulin knockdown. Strikingly, stable knockdown of βIII-tubulin significantly delayed tumor growth and reduced tumor incidence of subcutaneous xenografted tumors. The reduced growth and incidence of the tumors was not due to reduced cell proliferation, as stable knockdown of βIII-tubulin did not affect cell proliferation in vitro (Supplementary Fig. S7), suggesting that other factors associated with βIII-tubulin were at play. In the tumor environment, cancer cells survive under stressful conditions, and our finding that βIII-tubulin is a cellular survival factor for drug-induced cell death may extend to other known cellular stressors, including hypoxia and cytokine exposure (21). Consequently, there may be a balance between cell proliferation and cell death during tumor growth in our model. Alternatively, in the tumor microenvironment, the βIII-tubulin knockdown cells are proliferating at a slower rate. We support the former possibility as, once the tumors reached 1 cm3, staining with a proliferation marker (Ki67) did not reveal any difference in tumor cell proliferation in βIII-tubulin knockdown cells (Supplementary Fig. S8). Further investigation of cell proliferation and cell death parameters is under way to identify the cellular events mediated by βIII-tubulin during the early stages of in vivo tumor growth. The ability of βIII-tubulin knockdown to markedly suppress tumor progression strongly suggests an important role for this protein in lung cancer growth.
Our finding that βIII-tubulin is associated with tumorigenesis is not without precedent for a microtubule-related protein. For example, the microtubule-destabilizing protein stathmin has been linked with tumorigenesis in breast cancer and hepatocellular carcinoma (22–24). Moreover, a mutation in stathmin identified in esophageal adenocarcinoma was found to play a role in tumorigenesis (25). Despite the fact that βIII-tubulin is present in relatively small amounts in the NSCLC H460 cells (7.8% of total β-tubulin isotypes; ref. 11), knockdown of this minor isotype produces marked effects in drug response and tumorigenesis, suggesting that βIII-tubulin is functionally important in NSCLC cells. Our data strongly support a mechanistic role for βIII-tubulin in tumor cell behavior.
This study has shown that βIII-tubulin is a multifunctional protein that has a key role in the pathobiology and aggressiveness of human lung cancer by influencing drug sensitivity, tumor incidence, and progression. Our findings have direct clinical relevance and raise the possibility that future therapeutic strategies aimed at specifically blocking βIII-tubulin activity may have the dual advantage of suppressing lung cancer growth while enhancing the chemosensitivity of the tumor cells.
Disclosure of Potential Conflicts of Interest
J.A. McCarroll, P.P. Gan, and M. Kavallaris: Australian Provisional Application (No. PCT/AU2008/000298). Australian Provisional Applications: 2007901131/2007905307. Filed March 2007. Publication Number: WO08106730. Modulation of β-tubulin expression in tumour cells. M. Liu disclosed no potential conflicts of interest.
Grant Support: Children's Cancer Institute Australia for Medical Research, which is affiliated with the University of New South Wales and the Sydney Children's Hospital; National Health and Medical Research Council (M. Kavallaris); New South Wales Cancer Council (M. Kavallaris); University of New South Wales Faculty of Medicine Fellowship (J.A. McCarroll); University of New South Wales Faculty of Medicine Early Career Research Award (J.A. McCarroll); Endeavour International Postgraduate Research Scholarship (P.P. Gan); and National Health and Medical Research Council Senior Research Fellowship (M. Kavallaris).
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
- Received December 10, 2009.
- Revision received April 12, 2010.
- Accepted April 14, 2010.
- ©2010 American Association for Cancer Research.