Tumor Biol. DOI 10.1007/s13277-015-3361-y
RESEARCH ARTICLE
Overexpression of stathmin is resistant to paclitaxel treatment in patients with non-small cell lung cancer Ruifang Sun 1 & Zhigang Liu 2 & Lumin Wang 3 & Weidong Lv 2 & Jia Liu 2 & Caixia Ding 4 & Yong Yuan 4 & Guangyan Lei 2 & Changfu Xu 1
Received: 12 November 2014 / Accepted: 19 March 2015 # International Society of Oncology and BioMarkers (ISOBM) 2015
Abstract Paclitaxel can exert therapeutic effects by interacting with microtubules. Stathmin and β-III-tubulin, which have impact on microtubule activity, are believed to be involved in the chemotherapy. The purpose of the present study was to evaluate the associations between stathmin and β-III-tubulin expression and treatment response and survivals in patients with non-small cell lung cancer (NSCLC). Two hundred thirty-eight patients who were treated by platinumbased chemotherapy were enrolled in this study, among them, 111 patients also received paclitaxel treatment. Formalin-fixed and paraffin-embedded tumor tissues were collected for messenger RNA (mRNA) and protein detection. We assessed the associations of the two molecules with treatment response and survival outcome. High level of stathmin exhibited poor response to chemotherapy (for mRNA, P=0.041; for protein, P=0.017). Overexpression of stathmin was associated with shorter overall survival (for mRNA, P=0.012; for protein, P=0.014) and progression-free survival (for mRNA, P= Ruifang Sun and Zhigang Liu contributed equally to this work. * Ruifang Sun [email protected] 1
Department of Pathology, School of Medicine, Xi’an Jiaotong University, 76 Yanta West Road, Xi’an 710061, Shaanxi, People’s Republic of China
2
Department of Thoracic Surgery, Tumor Hospital of Shaanxi Province, 309 Yanta West Road, Xi’an, Shaanxi, People’s Republic of China
3
Key Laboratory of Environment and Genes Related to Diseases, School of Medicine, Xi’an Jiaotong University, 76 Yanta West Road, Xi’an, Shaanxi, People’s Republic of China
4
Department of Pathology, Tumor Hospital of Shaanxi Province, 309 Yanta West Road, Xi’an, Shaanxi, People’s Republic of China
0.039; for protein, P=0.022). Of note, this association was only observed in patients who were treated by both platinum and paclitaxel. Similar effects were not observed for β-IIItubulin. The findings demonstrated that paclitaxel effect may be interfered with stathmin; overexpression of stathmin is a predictive marker for a worse prognosis in patients with NSCLC who were treated by both platinum and paclitaxel chemotherapy.
Keywords STMN1 . Sensitivity . Prognosis . NSCLC . TAX . Chemotherapy
Introduction In spite of current improvements of diagnosis and treatment, lung cancer remains one of the leading causes of cancer mortality in both males and females worldwide [1]. It is reported that there are about 1.4 million new cases diagnosed with lung cancer every year [2]. Non-small cell lung cancer (NSCLC) accounts for approximately 85 % of all primary lung cancer cases, and most of them have advanced local invasion and/or distant metastases at the time of diagnosis. Although three therapeutic modalities including surgical resection, chemotherapy, and radiotherapy have been established in clinical application, the long-term survival of lung cancer is still generally poor; it is estimated that 5-year survival rate is only approximately 16 % [1, 3] which has not changed appreciably over the last two decades, and the recurrence rates remain high [4, 5]. Numerous clinical researches have demonstrated that chemotherapy is the main treatment choice for advanced NSCLC, which prolongs the survival of NSCLC patients [3]. Among various cytotoxic drugs in lung cancer clinical treatment, platinum is the most extensively used chemotherapeutic agent, and
Tumor Biol.
platinum-based double drug combination, platinum combined with vinblastine, paclitaxel, or gemcitabine has been recommended by 2011 guidelines of the National Comprehensive Cancer Network (NCCN) as a standard treatment for patients with a good performance status scale (PS) [6, 7]. However, NSCLC with the similar clinical pathology characteristics exhibits considerable heterogeneity in its sensitivity to chemotherapy [8]. Recently, individual treatment by selecting patients likely to respond to particular chemotherapeutic regimen is recommended by clinicians, which could improve the treatment efficacy and reduce the unnecessary side effects. Specific tumor biomarkers for chemotherapy treatment decision and prognosis are needed in clinics [6, 9]. However, very few biomarkers can be used for chemotherapy treatment decision or prognosis of lung cancer; thus, identification of such biomarkers is highly anticipated. Microtubules are dynamic α/β-tubulin heterodimers which play key roles in cell division, cell shape maintenance, cellular motility, and intracellular transport [10]. Numerous studies have demonstrated that microtubules are the target sites for many chemotherapy drugs. Paclitaxel (TAX), as an antimicrotubule, is widely used in the NSCLC chemotherapy as an antimicrotubule agent [11]. TAX can bind and stabilize β-tubulin in microtubules and prohibit depolymerization back to tubulin, leading to mitotic inhibition, such a function results in cell cycle arrest in G2/M phase and induces apoptosis [12]. Unfortunately, the resistance to TAX occurs frequently. β-tubulin has seven species of isotypes, higher level of expression of 1 isotype, tubulin, beta 3 Class III (β-III-tubulin), has been associated with chemotherapeutic resistance to the microtubule-binding protein; however, the results are inconsistent; some findings supported the association, while some others failed to find such an association [13]. Stathmin, also known as stathmin 1 (STMN1), p17, p18, p19, 19 K, metablastin, LAP18, and oncoprotein 18 (Op18), encodes a 19-kDa cytosolic protein consists of 149 amino acids, and the core region (amino acids 42–126) is the minimum fragment that required for tubulin interaction [14, 15]. Stathmin can modulate microtubule dynamics through preventing polymerization of tubulin and boosting destabilization and disassembly of microtubule during the interphase and late mitosis along cell cycle progression; this process is regulated by changes in the phosphorylation status of stathmin [16]. Stathmin also plays roles in a variety of other biological processes, such as cell proliferation, mobility, metastasis, differentiation, and resisting to antimicrotubule therapy [16–18]. Thus, we suppose that the influence of TAX on microtubule polymerization maybe varied by different expression level of stathmin [19]. Currently, a study showed that overexpression of stathmin is a poor prognostic biomarker in non-small cell lung cancer. However, whether the difference between stathmin expression levels and survival were determined by TAX treatment was not investigated by that study [20].
Thus, the aim of the present study was to evaluate whether β-III-tubulin or stathmin can be a predictive marker for the choice of chemotherapy regimen with or without TAX. Therefore, NSCLC patients who were treated by platinumbased chemotherapy combined with or without TAX were enrolled in our study; we evaluated the associations between β-III-tubulin and stathmin expression (both in messenger RNA (mRNA) and protein levels) and NSCLC survival (including overall and progression-free survival), as well as response to platinum-based chemotherapy.
Materials and methods Population A total of 238 patients diagnosed with stage III or IV (either de novo or relapsed) NSCLC who have not undertaken lobectomy resection but received platinum-based chemotherapy in Shaan’Xi Tumor Hospital between October 2007 and February 2011 were enrolled in the present study. The collected biopsy specimens were made into formalin-fixed and paraffin-embedded (FFPE) tissues for pathological diagnosis and immunohistochemical staining. Each tumor sample made into paraffin slide was reviewed by two independent pathologists and assigned a histological grade and disease stage based on the World Health Organization classification criteria and the seventh edition of the International Union Against Cancer Tumor Node Metastasis (TNM) staging system. The demographic and clinical information were collected from medical records and described in Table 1. The patients were treated by platinum-based chemotherapy combined with or without TAX. Tumor response to chemotherapy was assessed after two treatment cycles by clinical test, imaging examination, and serum CA-125 according to Response Evaluation Criteria in Solid Tumors (RECIST) 1.1 criteria. In order to get the result of the analysis, patients who had a stable disease (SD) and progression disease (PD) were defined as non-responders and patients who had a complete response (CR) and a partial response (PR) were considered as responders. Of the 238 patients treated by platinum-based chemotherapy, 108 (45.30 %) patients were responders and 130 (54.62 %) were non-responders. All the patients had well-documented clinical histories and follow-up information which was obtained through office visits or telephone interviews, either with the patient or with a relative. Patients were followed up from chemotherapy through February 2011 for clinical outcome. The median overall survival (OS) from the date of chemotherapy to the date of either death or last contact was 7.8 months (range, 3– 48 months). The median disease progression-free survival (PFS), which were considered as the time from the date of chemotherapy to the date of first local relapse, distant
Tumor Biol. Table 1
The main clinical characteristics of patients
Characteristic
Patients (n=238)
Percentage
Age (range), year Mean ≤60 >60 Gender
(25–87) 59.67 126 112
52.94 47.06
Male Female Smoking status Never Current or ever Pathological type Squamous cell carcinoma Adenocarcinoma Tumor differentiation Well Moderate Poor Stage III IV Chemotherapy Platinum-based with TAX Platinum-based without TAX
162 76
68.07 31.93
107 131
44.96 55.04
91 147
38.24 61.76
19 101 118
7.98 42.44 49.58
93 145
39.08 60.92
111 127
46.64 53.36
108 130
45.38 54.62
Response to chemotherapy Responder (CR+PR) Non-responder (SD+PD)
TAX paclitaxel, CR complete response, PR partial response, SD stable disease, PD progression disease
metastasis, death without relapse, or last follow-up, was 5 months (range, 1.1–20 months). The study is reviewed and approved by Ethics Committee of Medical College of Xi’an Jiaotong University, Xi’an, China, and all participants provided consent for the sample collection and data analysis.
sequences were AAA TGG CTG CCA AAC TGG AAC GT (stathmin forward), GCT TCA GTC TCG TCA GCA GGG TC (stathmin reverse), ACA GCA GCT ACT TCG TGG AGT GGA TC (β-III-tubulin forward), GTC TTC GTA CAT CTC GCC CTC TTC C (β-III-tubulin reverse), GAA GGT GAA GGT CGG AGT C (GAPDH forward), and GAA GAT GGT GAT GGG ATT TC (GAPDH reverse), respectively, as previously described [21]. All experiments were ran in triplicate, and water blanks were included as negative controls. The relative quantification of stathmin and β-III-tubulin mRNA expression was calculated using standard ΔΔCt method. For categorical analysis, the expression level was divided into low and high groups according to median distribution. Immunohistochemistry Stathmin and β-III-tubulin protein expression were also investigated by immunohistochemistry analysis. In brief, FFPE tissue sections (5-μm thick) were deparaffinized, rehydrated, endogenous-peroxide-blocked, and antigen-retrieved sequentially. Then, the sections were incubated with stathmin polyclonal antibody (Cell Signaling Technology, Boston, MA, USA) and β-III-tubulin monoclonal antibody (Cell Signaling Technology, Boston, MA, USA) at 1:50 and 1:100 dilution, respectively, and overnight at 4 °C. For negative controls, phosphate-buffered saline (PBS) was used in place of primary antibody. Then, the tissue sections were washed with PBS and followed by incubation with HRP-conjugated secondary antibody (Maixin Biological, Fuzhou, China) for 30 min. Finally, the sections were developed with 3,3′-diaminobenzidine (DAB) solution, and all of the slides were counterstained with hematoxylin, dehydrated with ethanol, cleaned with xylene, and mounted on cover slips. The degree of immunostaining was evaluated by two independent pathologists who were blind to the clinical and follow-up data of the patients. Each slide was evaluated under five random fields at ×400 magnification, and 100 cells were counted. We used a scoring standard for stathmin and β-III-tubulin protein expression, and both the percentage of positive cells and intensity were considered as previously described [22, 23].
Extraction of total RNA and real-time quantitative PCR Statistical analysis Total RNA was extracted from FFPE tissues using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). RNA concentration was measured by NanoDrop 1000 (Thermo Fisher Scientific, Waltham, MA, USA). RNA was reverse transcribed to cDNA using PrimeScript RT reagent kit (TaKaRa, Dalian, China) according to the manufacturer's instructions. Quantitative realtime PCR (qRT-PCR) was performed to detect levels of stathmin and β-III-tubulin mRNA expression using an ABI Prism 7000 Sequence Detection System (Life Technologies, Carlsbad, CA, USA). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as the internal control. The primer
Continuous and categorical variables were expressed as mean ± standard deviation and n of subjects (percentage). The association between stathmin and β-III-tubulin expression and clinicopathologic factors and response to chemotherapy were assessed by Pearson’s chi-squared test and the Mann– Whitney U test. Survival time was calculated, and patients who were alive at the last follow-up were censored. Survival curves were plotted by the Kaplan–Meier method, and logrank test was applied to compare survival rates between subgroups. All statistical analyses were conducted using SPSS
Tumor Biol.
17.0 software (SPSS, IBM, Chicago, IL). All statistical tests were two sided and P value less than 0.05 was considered statistically significant.
Results Clinical characteristics and outcome of NSCLC patients The demographic and clinical characteristics of the 238 enrolled patients with NSCLC in the present study are summarized in Table 1. The patients (162 men and 76 women) were 25–87 years old at diagnosis; the average age was 59.67 years. Among these patients, 91 (38.24 %) were diagnosed with squamous cell carcinomas and 147 (61.76 %) were diagnosed with adenocarcinomas. Correlation between stathmin and β-III-tubulin mRNA expression and clinicopathological parameters in NSCLC The mRNA levels of stathmin and β-III-tubulin were determined with qRT-PCR assays in 238 patients with NSCLC. Clinicopathological characteristics of NSCLC are classified Table 2 Association of mRNA expression levels of stathmin and β-IIItubulin with clinical features
according to stathmin and β-III-tubulin mRNA expression status, respectively (Table 2). Both of the stathmin and β-IIItubulin mRNA expression levels were not correlated with the clinical features including gender, age, smoking status, pathological type, tumor differentiation, stage, and chemotherapy, respectively (P>0.05). Of note, patients with high level of stathmin expression tended to exhibit poor response to chemotherapy (P=0.041). Correlation between stathmin and β-III-tubulin protein expression and clinicopathological parameters in NSCLC For the purpose of confirming the molecular biological findings and evaluating the clinicopathological and prognostic roles of stathmin and β-III-tubulin expression, we conducted IHC analysis in paraffin-embedded NSCLC sections. Among the 238 NSCLC samples, high stathmin and β-III-tubulin expression accounted for 128 (53.78 %) and 189 (79.41 %), whereas the remaining 110 cases (46.22 %) of stathmin and 49 cases (20.59 %) of β-III-tubulin displayed low expression levels (Table 3). The relationship between stathmin and β-IIItubulin expression and other clinicopathological features of NSCLC were showed in Table 3. High expression of stathmin
Characteristic
All patients (n=238)
Stathmin expression Median (range)
P
β-III-tubulin expression Median (range)
P
Age (range), year ≤60 >60 Gender Male Female Smoking status Never Current or ever Pathological type Squamous cell carcinoma Adenocarcinoma Tumor differentiation Well Moderate Poor
238 126 112
1.53 (0.06–197.10) 4.32 (0.09–197.10) 1.22 (0.06–186.73)
0.144
0.33 (0–25.13) 0.54 (0–25.13) 0.08 (0–24.85)
0.603
162 76
1.46 (0.06–197.10) 1.57 (0.11–188.28)
0.269
0.33 (0–24.75) 0.08 (0–25.13)
0.885
107 131
1.33 (0.06–188.28) 2.24 (0.08–197.10)
0.578
0.08 (0–25.13) 0.33 (0–24.88)
0.722
91 147
4.34 (0.08–76.05) 1.33 (0.06–197.10)
0.059
0.43 (0.01–24.75) 0.25 (0–25.13)
0.879
19 101 118
2.72 (0.47–77.24) 4.48 (0.08–188.28) 1.27 (0.06–197.10)
0.361
0.53 (0–22.23) 0.79 (0–25.13) 0.25 (0–24.88)
0.327
93 145
2.72 (0.08–188.28) 1.5 (0.06–197.10)
0.957
0.32 (0–24.88) 0.08 (0–25.13)
0.059
111 127
4.18 (0.11–177.49) 1.29 (0.06–197.10)
0.321
0.25 (0–25.13) 0.33 (0–24.85)
0.712
108 130
1.22 (0.08–76.05) 4.48 (0.06–197.10)
0.041
0.33 (0–24.88) 0.18 (0–25.13)
0.993
Stage III IV Chemotherapy Platinum-based with TAX Platinum-based without TAX Response to chemotherapy Responder (CR+PR) Non-responder (SD+PD)
TAX paclitaxel, CR complete response, PR partial response, SD stable disease, PD progression disease
Tumor Biol. Table 3 Association of protein expression levels of stathmin and β-IIItubulin with clinical features
Characteristic
All patients
Stathmin expression
(n=238)
Low (n)
High (n)
126 112
56 54
70 58
162 76
79 31
107 131
P
β-III-tubulin expression
P
Low (n)
High (n)
0.560
25 24
101 88
0.762
83 45
0.250
36 13
126 63
0.363
46 64
61 67
0.367
21 28
86 103
0.740
91 147
40 70
51 77
0.582
18 31
73 116
0.808
19 101 118
12 54 46
7 47 72
0.035
5 20 24
14 81 94
93 145
45 67
48 78
0.742
19 30
74 115
0.961
127 111
66 46
61 65
0.105
27 22
100 89
0.784
108 130
60 52
48 78
0.017
24 25
84 105
0.570
Age (range), year ≤60 >60 Gender Male Female Smoking status Never Current or ever Pathological type Squamous cell carcinoma Adenocarcinoma Tumor differentiation Well Moderate Poor Stage III IV Chemotherapy Platinum-based with TAX Platinum-based without TAX Response to chemotherapy Responder (CR+PR) Non-responder (SD+PD)
TAX paclitaxel, CR complete response, PR partial response, SD stable disease, PD progression disease
was tended to be poor differentiation (P=0.035) and poor response to chemotherapy (P=0.017). However, no significant relationship was found between β-III-tubulin expression and any of the listed clinical features (P>0.05).
Correlation of stathmin and β-III-tubulin mRNA expression with survivals The OS and PFS using the Kaplan–Meier analysis revealed that the prognosis of NSCLC patients with high stathmin mRNA expression was significantly poorer than those with low stathmin expression (for OS: log-rank P =0.012; for PFS: log-rank P=0.039). Moreover, stratified analysis by treatment indicated that the associations between stathmin expression level and OS and PFS were substantial in patients who were treated by both platinum and TAX (for OS: log-rank P = 0.010; for PFS: log-rank P = 0.031, respectively). Similarity in survival differences were not found for patients who were treated by platinum without TAX (for OS: log-rank P=0.367; for PFS: log-rank P=0.457, respectively) (Figs. 1 and 2).
As for β-III-tubulin, Kaplan–Meier plots showed that β-IIItubulin expression was tended to be associated with PFS but not OS. When compared to low expression, patients with higher expression level of β-III-tubulin was slightly correlated with shorter PFS (log-rank P=0.163). The results stratified by treatment regimens indicated that the slight correlation between β-IIItubulin expression and PFS was significant only among patients who were treated by both platinum and TAX (log-rank P=0.038). Similar PFS difference was not found for patients who received both platinum without TAX (log-rank P=0.859) (Fig. 3). Correlation of stathmin and β-III-tubulin protein expression with survivals The correlation of stathmin and β-III-tubulin protein expression with survivals and response were consistent with the correlation of their mRNA expression with survivals and response. In brief, high stathmin protein expression were significantly associated with shorter OS and PFS than low stathmin expression (for OS: log-rank P=0.014; for PFS: log-rank P= 0.022). Moreover, the associations were still significant only among patients who were treated by both platinum and TAX
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Fig. 1 Kaplan–Meier curves for overall survival according to stathmin expression in patients with NSCLC. a Overall survival according to stathmin mRNA level in patients who were treated by platinum-based chemotherapy without paclitaxel. b Overall survival according to stathmin mRNA level in patients who were treated by platinum-based
chemotherapy combined with paclitaxel. c Overall survival according to stathmin protein level in patients who were treated by platinum-based chemotherapy without paclitaxel. d Overall survival according to stathmin protein level in patients who were treated by platinum-based chemotherapy combined with paclitaxel
(for OS: log-rank P=0.005; for PFS: log-rank P=0.016, respectively). Similar differences of survival were not found for patients who were treated by platinum-based chemotherapy without TAX (for OS: log-rank P=0.626; for PFS: log-rank P=0.422, respectively) (Figs. 1 and 2). Patients with higher β-III-tubulin expression were slightly correlated with shorter PFS (log-rank P=0.078) when compared to lower expression. Stratified analysis showed that the correlation between β-III-tubulin expression and PFS was significant only among patients who were treated by platinumbased chemotherapy without TAX (log-rank P=0.094) (Fig. 3).
high. The limited efficacy of cytotoxic chemotherapy and anticancer drug resistance are major obstacles for the treatment of advanced lung cancer patients [24]. Antimicrotubule agents are widely used chemotherapy drugs for lung cancer treatment [25]. Currently, the relationship between microtubule dynamics and TAX resistance attracted more and more attention; therefore, β-IIItubulin and stathmin which can interact with microtubules become research hotspots. β-III-tubulin has been associated with chemotherapeutic resistance to the microtubulebinding protein; however, the results are inconsistent [13]. Previous studies in endometrial and lung cancer cell lines showed that increased levels of stathmin were resistant to TAX [17, 26]; however, the role of stathmin expression in TAX resistance was detected by few studies in vivo. In our present study, we investigated the relationship between β-III-tubulin, stathmin expression (both in mRNA
Discussion There are significant advances in oncology in recent decades, even though the mortality of lung cancer remains
Tumor Biol.
Fig. 2 Kaplan–Meier curves for progression-free survival according to stathmin expression in patients with NSCLC. a Progression-free survival according to stathmin mRNA level in patients who were treated by platinum-based chemotherapy without paclitaxel. b Progression-free survival according to stathmin mRNA level in patients who were treated by platinum-based chemotherapy combined with paclitaxel. c
Progression-free survival according to stathmin protein level in patients who were treated by platinum-based chemotherapy without paclitaxel. d Progression-free survival according to stathmin protein level in patients who were treated by platinum-based chemotherapy combined with paclitaxel
and protein levels), and clinical outcome in NSCLC patients who received platinum-based chemotherapy with and without TAX. Our results showed that stathmin expression (both in mRNA and protein levels) was not only associated with PFS and OS but also was related to chemotherapy response. However, βIII-tubulin expression (both in mRNA and protein levels) was only associated with PFS but not OS; besides, β-III-tubulin expression was not correlated to chemotherapy response. Of note, the associations were substantial only in patients who were treated by both platinum and TAX, but not in patients who were treated by platinum without TAX. These findings suggest that the expression level of stathmin (or β-III-tubulin) has an impact on the therapeutic effect of TAX. Stathmin, which is considered as a microtubuledestabilizing protein, can modulate microtubule dynamics
through preventing polymerization of tubulin and boosting destabilization and disassembly of microtubule during cell cycle progression [16]. Hence, elevated expression level of stathmin may play a role in disturbing microtubule stabilization; therefore, the influence of TAX on microtubule polymerization may be affected. Besides, stathmin is implicated in the regulation process of cell proliferation, mobility, metastasis, and differentiation, which may also have an impact on tumor progression [16, 19]. Our results suggest that stathmin mRNA or protein expression level can be considered as a strong predictive marker of NSCLC survival after platinum and TAX chemotherapy. There are several possible mechanisms to elucidate our findings; TAX resistance is considered as one of the probabilities. It was demonstrated by a previous study that targeting stathmin and Bcl-2 expression with RNA interference (RNAi) significantly downregulates RNA and protein
Tumor Biol.
Fig. 3 Kaplan–Meier curves for progression-free survival according to β-III-tubulin expression in patients with NSCLC. a Progression-free survival according to β-III-tubulin mRNA level in patients who were treated by platinum-based chemotherapy without paclitaxel. b Progression-free survival according to β-III-tubulin mRNA level in patients who were treated by platinum-based chemotherapy combined
with paclitaxel. c Progression-free survival according to β-III-tubulin protein level in patients who were treated by platinum-based chemotherapy without paclitaxel. d Progression-free survival according to β-III-tubulin protein level patients who were treated by platinum-based chemotherapy combined with paclitaxel
expression levels of stathmin and Bcl-2 and sensitizes lung cancer cells to TAX [26]. In addition, in breast cancer cell lines, higher expression level of stathmin diminished microtubule polymerization, leading to cell cycle arrest at G2 phase, thus evidently decreased the binding of TAX to microtubules [27]; on the contrary, stathmin suppression by RNAi promoted microtubule polymerization and facilitated cell cycle progression from G2 to M phase; therefore, cell sensitivity to TAX was elevated [28]. Besides, two TAX-resistant ovarian cancer cell lines exhibit higher expression level of stathmin [29]. Furthermore, in prostate cancer cell lines, stathmin inhibition and TAX exposure had a synergistic activity on proliferation and angiogenesis [30]. In summary, all the above findings indicate that stathmin may interfere with tumor response to TAX treatment. The association between stathmin expression (both in mRNA and protein levels) and NSCLC
prognosis in our present study becomes another evidence that stathmin may be implicated in lung cancer resistance to TAX treatment. In our current study, β-III-tubulin expression was associated with PFS but was not related to chemotherapy response. As for this result, we need to give comprehensive explanations. To our knowledge, there is a supposition that the prognosis of patients will be better if they initially respond well to chemotherapy; therefore, the response to treatment has become an early predictive marker [31]. However, patients who have comparable response to initial treatment can result in different clinical outcomes. The associations between treatment response and survival are different in different studies because of inconsistent treatment protocols and evaluation criterion of response. Of note, in our study, response to treatment was assessed after two treatment cycles. There is a possibility that
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the treatment response was evaluated too soon; thus far, the correction between response and prognosis was not observed. In addition, although the results showed that β-III-tubulin was related to PFS but not OS, the complication of clinical and pathological differentiation among lung cancer patients may also have resulted in the inability to investigate the relations between β-III-tubulin and treatment response. Interestingly, the results also showed that the expression of β-III-tubulin (both in mRNA and protein levels) tended to be associated with NSCLC survival, but the relation was only observed in progression-free survival among patients who were treated by platinum-based chemotherapy without TAX. We need to consider the data carefully. Previous studies in vitro have suggested that overexpression of β-III-tubulin can lead to TAX resistance through decreasing microtubule assembly and reducing the ability of TAX to prohibit microtubule dynamics [32]. Results from previous studies also demonstrated associations between higher level of β-III-tubulin and TAX resistance in patients with ovarian cancer [21], breast cancer [33], and NSCLC [34], which are consistent with our results. Although previous studies also showed inconsistent results [13], combined with our results, we considered that platinum-based chemotherapy with TAX regimen may be a little more beneficial for patient with NSCLC, compared with platinum-based chemotherapy without TAX regimen. Further investigations should be carried out on the predictive value of βIII-tubulin expression in TAX resistance, chemotherapy response, and survival of a larger and independent cohort in the future. In summary, although the underlying mechanism remains to be elucidated, the results showed that stathmin expressions (both in mRNA and protein levels) are important predictive markers for NSCLC patients who are treated by platinum-based chemotherapy combined with TAX. Besides, overexpression of stathmin was significantly associated with shorter progression-free survival and overall survival. Our findings demonstrate that stathmin may play a role in interfering the platinum and TAX treatment on NSCLC patients, and expression of stathmin should be investigated in TAX treatment in patients with NSCLC in further studies.
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Acknowledgments This work was partly supported by Postdoctoral Science Foundation of China (No. 2014M552461). We would like to thank all the participants and staff of Shaan’Xi Tumor Hospital for their valuable contributions on collecting specimens and sorting out clinical data. Conflicts of interest None
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RESEARCH ARTICLE
Overexpression of stathmin is resistant to paclitaxel treatment in patients with non-small cell lung cancer Ruifang Sun 1 & Zhigang Liu 2 & Lumin Wang 3 & Weidong Lv 2 & Jia Liu 2 & Caixia Ding 4 & Yong Yuan 4 & Guangyan Lei 2 & Changfu Xu 1
Received: 12 November 2014 / Accepted: 19 March 2015 # International Society of Oncology and BioMarkers (ISOBM) 2015
Abstract Paclitaxel can exert therapeutic effects by interacting with microtubules. Stathmin and β-III-tubulin, which have impact on microtubule activity, are believed to be involved in the chemotherapy. The purpose of the present study was to evaluate the associations between stathmin and β-III-tubulin expression and treatment response and survivals in patients with non-small cell lung cancer (NSCLC). Two hundred thirty-eight patients who were treated by platinumbased chemotherapy were enrolled in this study, among them, 111 patients also received paclitaxel treatment. Formalin-fixed and paraffin-embedded tumor tissues were collected for messenger RNA (mRNA) and protein detection. We assessed the associations of the two molecules with treatment response and survival outcome. High level of stathmin exhibited poor response to chemotherapy (for mRNA, P=0.041; for protein, P=0.017). Overexpression of stathmin was associated with shorter overall survival (for mRNA, P=0.012; for protein, P=0.014) and progression-free survival (for mRNA, P= Ruifang Sun and Zhigang Liu contributed equally to this work. * Ruifang Sun [email protected] 1
Department of Pathology, School of Medicine, Xi’an Jiaotong University, 76 Yanta West Road, Xi’an 710061, Shaanxi, People’s Republic of China
2
Department of Thoracic Surgery, Tumor Hospital of Shaanxi Province, 309 Yanta West Road, Xi’an, Shaanxi, People’s Republic of China
3
Key Laboratory of Environment and Genes Related to Diseases, School of Medicine, Xi’an Jiaotong University, 76 Yanta West Road, Xi’an, Shaanxi, People’s Republic of China
4
Department of Pathology, Tumor Hospital of Shaanxi Province, 309 Yanta West Road, Xi’an, Shaanxi, People’s Republic of China
0.039; for protein, P=0.022). Of note, this association was only observed in patients who were treated by both platinum and paclitaxel. Similar effects were not observed for β-IIItubulin. The findings demonstrated that paclitaxel effect may be interfered with stathmin; overexpression of stathmin is a predictive marker for a worse prognosis in patients with NSCLC who were treated by both platinum and paclitaxel chemotherapy.
Keywords STMN1 . Sensitivity . Prognosis . NSCLC . TAX . Chemotherapy
Introduction In spite of current improvements of diagnosis and treatment, lung cancer remains one of the leading causes of cancer mortality in both males and females worldwide [1]. It is reported that there are about 1.4 million new cases diagnosed with lung cancer every year [2]. Non-small cell lung cancer (NSCLC) accounts for approximately 85 % of all primary lung cancer cases, and most of them have advanced local invasion and/or distant metastases at the time of diagnosis. Although three therapeutic modalities including surgical resection, chemotherapy, and radiotherapy have been established in clinical application, the long-term survival of lung cancer is still generally poor; it is estimated that 5-year survival rate is only approximately 16 % [1, 3] which has not changed appreciably over the last two decades, and the recurrence rates remain high [4, 5]. Numerous clinical researches have demonstrated that chemotherapy is the main treatment choice for advanced NSCLC, which prolongs the survival of NSCLC patients [3]. Among various cytotoxic drugs in lung cancer clinical treatment, platinum is the most extensively used chemotherapeutic agent, and
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platinum-based double drug combination, platinum combined with vinblastine, paclitaxel, or gemcitabine has been recommended by 2011 guidelines of the National Comprehensive Cancer Network (NCCN) as a standard treatment for patients with a good performance status scale (PS) [6, 7]. However, NSCLC with the similar clinical pathology characteristics exhibits considerable heterogeneity in its sensitivity to chemotherapy [8]. Recently, individual treatment by selecting patients likely to respond to particular chemotherapeutic regimen is recommended by clinicians, which could improve the treatment efficacy and reduce the unnecessary side effects. Specific tumor biomarkers for chemotherapy treatment decision and prognosis are needed in clinics [6, 9]. However, very few biomarkers can be used for chemotherapy treatment decision or prognosis of lung cancer; thus, identification of such biomarkers is highly anticipated. Microtubules are dynamic α/β-tubulin heterodimers which play key roles in cell division, cell shape maintenance, cellular motility, and intracellular transport [10]. Numerous studies have demonstrated that microtubules are the target sites for many chemotherapy drugs. Paclitaxel (TAX), as an antimicrotubule, is widely used in the NSCLC chemotherapy as an antimicrotubule agent [11]. TAX can bind and stabilize β-tubulin in microtubules and prohibit depolymerization back to tubulin, leading to mitotic inhibition, such a function results in cell cycle arrest in G2/M phase and induces apoptosis [12]. Unfortunately, the resistance to TAX occurs frequently. β-tubulin has seven species of isotypes, higher level of expression of 1 isotype, tubulin, beta 3 Class III (β-III-tubulin), has been associated with chemotherapeutic resistance to the microtubule-binding protein; however, the results are inconsistent; some findings supported the association, while some others failed to find such an association [13]. Stathmin, also known as stathmin 1 (STMN1), p17, p18, p19, 19 K, metablastin, LAP18, and oncoprotein 18 (Op18), encodes a 19-kDa cytosolic protein consists of 149 amino acids, and the core region (amino acids 42–126) is the minimum fragment that required for tubulin interaction [14, 15]. Stathmin can modulate microtubule dynamics through preventing polymerization of tubulin and boosting destabilization and disassembly of microtubule during the interphase and late mitosis along cell cycle progression; this process is regulated by changes in the phosphorylation status of stathmin [16]. Stathmin also plays roles in a variety of other biological processes, such as cell proliferation, mobility, metastasis, differentiation, and resisting to antimicrotubule therapy [16–18]. Thus, we suppose that the influence of TAX on microtubule polymerization maybe varied by different expression level of stathmin [19]. Currently, a study showed that overexpression of stathmin is a poor prognostic biomarker in non-small cell lung cancer. However, whether the difference between stathmin expression levels and survival were determined by TAX treatment was not investigated by that study [20].
Thus, the aim of the present study was to evaluate whether β-III-tubulin or stathmin can be a predictive marker for the choice of chemotherapy regimen with or without TAX. Therefore, NSCLC patients who were treated by platinumbased chemotherapy combined with or without TAX were enrolled in our study; we evaluated the associations between β-III-tubulin and stathmin expression (both in messenger RNA (mRNA) and protein levels) and NSCLC survival (including overall and progression-free survival), as well as response to platinum-based chemotherapy.
Materials and methods Population A total of 238 patients diagnosed with stage III or IV (either de novo or relapsed) NSCLC who have not undertaken lobectomy resection but received platinum-based chemotherapy in Shaan’Xi Tumor Hospital between October 2007 and February 2011 were enrolled in the present study. The collected biopsy specimens were made into formalin-fixed and paraffin-embedded (FFPE) tissues for pathological diagnosis and immunohistochemical staining. Each tumor sample made into paraffin slide was reviewed by two independent pathologists and assigned a histological grade and disease stage based on the World Health Organization classification criteria and the seventh edition of the International Union Against Cancer Tumor Node Metastasis (TNM) staging system. The demographic and clinical information were collected from medical records and described in Table 1. The patients were treated by platinum-based chemotherapy combined with or without TAX. Tumor response to chemotherapy was assessed after two treatment cycles by clinical test, imaging examination, and serum CA-125 according to Response Evaluation Criteria in Solid Tumors (RECIST) 1.1 criteria. In order to get the result of the analysis, patients who had a stable disease (SD) and progression disease (PD) were defined as non-responders and patients who had a complete response (CR) and a partial response (PR) were considered as responders. Of the 238 patients treated by platinum-based chemotherapy, 108 (45.30 %) patients were responders and 130 (54.62 %) were non-responders. All the patients had well-documented clinical histories and follow-up information which was obtained through office visits or telephone interviews, either with the patient or with a relative. Patients were followed up from chemotherapy through February 2011 for clinical outcome. The median overall survival (OS) from the date of chemotherapy to the date of either death or last contact was 7.8 months (range, 3– 48 months). The median disease progression-free survival (PFS), which were considered as the time from the date of chemotherapy to the date of first local relapse, distant
Tumor Biol. Table 1
The main clinical characteristics of patients
Characteristic
Patients (n=238)
Percentage
Age (range), year Mean ≤60 >60 Gender
(25–87) 59.67 126 112
52.94 47.06
Male Female Smoking status Never Current or ever Pathological type Squamous cell carcinoma Adenocarcinoma Tumor differentiation Well Moderate Poor Stage III IV Chemotherapy Platinum-based with TAX Platinum-based without TAX
162 76
68.07 31.93
107 131
44.96 55.04
91 147
38.24 61.76
19 101 118
7.98 42.44 49.58
93 145
39.08 60.92
111 127
46.64 53.36
108 130
45.38 54.62
Response to chemotherapy Responder (CR+PR) Non-responder (SD+PD)
TAX paclitaxel, CR complete response, PR partial response, SD stable disease, PD progression disease
metastasis, death without relapse, or last follow-up, was 5 months (range, 1.1–20 months). The study is reviewed and approved by Ethics Committee of Medical College of Xi’an Jiaotong University, Xi’an, China, and all participants provided consent for the sample collection and data analysis.
sequences were AAA TGG CTG CCA AAC TGG AAC GT (stathmin forward), GCT TCA GTC TCG TCA GCA GGG TC (stathmin reverse), ACA GCA GCT ACT TCG TGG AGT GGA TC (β-III-tubulin forward), GTC TTC GTA CAT CTC GCC CTC TTC C (β-III-tubulin reverse), GAA GGT GAA GGT CGG AGT C (GAPDH forward), and GAA GAT GGT GAT GGG ATT TC (GAPDH reverse), respectively, as previously described [21]. All experiments were ran in triplicate, and water blanks were included as negative controls. The relative quantification of stathmin and β-III-tubulin mRNA expression was calculated using standard ΔΔCt method. For categorical analysis, the expression level was divided into low and high groups according to median distribution. Immunohistochemistry Stathmin and β-III-tubulin protein expression were also investigated by immunohistochemistry analysis. In brief, FFPE tissue sections (5-μm thick) were deparaffinized, rehydrated, endogenous-peroxide-blocked, and antigen-retrieved sequentially. Then, the sections were incubated with stathmin polyclonal antibody (Cell Signaling Technology, Boston, MA, USA) and β-III-tubulin monoclonal antibody (Cell Signaling Technology, Boston, MA, USA) at 1:50 and 1:100 dilution, respectively, and overnight at 4 °C. For negative controls, phosphate-buffered saline (PBS) was used in place of primary antibody. Then, the tissue sections were washed with PBS and followed by incubation with HRP-conjugated secondary antibody (Maixin Biological, Fuzhou, China) for 30 min. Finally, the sections were developed with 3,3′-diaminobenzidine (DAB) solution, and all of the slides were counterstained with hematoxylin, dehydrated with ethanol, cleaned with xylene, and mounted on cover slips. The degree of immunostaining was evaluated by two independent pathologists who were blind to the clinical and follow-up data of the patients. Each slide was evaluated under five random fields at ×400 magnification, and 100 cells were counted. We used a scoring standard for stathmin and β-III-tubulin protein expression, and both the percentage of positive cells and intensity were considered as previously described [22, 23].
Extraction of total RNA and real-time quantitative PCR Statistical analysis Total RNA was extracted from FFPE tissues using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). RNA concentration was measured by NanoDrop 1000 (Thermo Fisher Scientific, Waltham, MA, USA). RNA was reverse transcribed to cDNA using PrimeScript RT reagent kit (TaKaRa, Dalian, China) according to the manufacturer's instructions. Quantitative realtime PCR (qRT-PCR) was performed to detect levels of stathmin and β-III-tubulin mRNA expression using an ABI Prism 7000 Sequence Detection System (Life Technologies, Carlsbad, CA, USA). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as the internal control. The primer
Continuous and categorical variables were expressed as mean ± standard deviation and n of subjects (percentage). The association between stathmin and β-III-tubulin expression and clinicopathologic factors and response to chemotherapy were assessed by Pearson’s chi-squared test and the Mann– Whitney U test. Survival time was calculated, and patients who were alive at the last follow-up were censored. Survival curves were plotted by the Kaplan–Meier method, and logrank test was applied to compare survival rates between subgroups. All statistical analyses were conducted using SPSS
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17.0 software (SPSS, IBM, Chicago, IL). All statistical tests were two sided and P value less than 0.05 was considered statistically significant.
Results Clinical characteristics and outcome of NSCLC patients The demographic and clinical characteristics of the 238 enrolled patients with NSCLC in the present study are summarized in Table 1. The patients (162 men and 76 women) were 25–87 years old at diagnosis; the average age was 59.67 years. Among these patients, 91 (38.24 %) were diagnosed with squamous cell carcinomas and 147 (61.76 %) were diagnosed with adenocarcinomas. Correlation between stathmin and β-III-tubulin mRNA expression and clinicopathological parameters in NSCLC The mRNA levels of stathmin and β-III-tubulin were determined with qRT-PCR assays in 238 patients with NSCLC. Clinicopathological characteristics of NSCLC are classified Table 2 Association of mRNA expression levels of stathmin and β-IIItubulin with clinical features
according to stathmin and β-III-tubulin mRNA expression status, respectively (Table 2). Both of the stathmin and β-IIItubulin mRNA expression levels were not correlated with the clinical features including gender, age, smoking status, pathological type, tumor differentiation, stage, and chemotherapy, respectively (P>0.05). Of note, patients with high level of stathmin expression tended to exhibit poor response to chemotherapy (P=0.041). Correlation between stathmin and β-III-tubulin protein expression and clinicopathological parameters in NSCLC For the purpose of confirming the molecular biological findings and evaluating the clinicopathological and prognostic roles of stathmin and β-III-tubulin expression, we conducted IHC analysis in paraffin-embedded NSCLC sections. Among the 238 NSCLC samples, high stathmin and β-III-tubulin expression accounted for 128 (53.78 %) and 189 (79.41 %), whereas the remaining 110 cases (46.22 %) of stathmin and 49 cases (20.59 %) of β-III-tubulin displayed low expression levels (Table 3). The relationship between stathmin and β-IIItubulin expression and other clinicopathological features of NSCLC were showed in Table 3. High expression of stathmin
Characteristic
All patients (n=238)
Stathmin expression Median (range)
P
β-III-tubulin expression Median (range)
P
Age (range), year ≤60 >60 Gender Male Female Smoking status Never Current or ever Pathological type Squamous cell carcinoma Adenocarcinoma Tumor differentiation Well Moderate Poor
238 126 112
1.53 (0.06–197.10) 4.32 (0.09–197.10) 1.22 (0.06–186.73)
0.144
0.33 (0–25.13) 0.54 (0–25.13) 0.08 (0–24.85)
0.603
162 76
1.46 (0.06–197.10) 1.57 (0.11–188.28)
0.269
0.33 (0–24.75) 0.08 (0–25.13)
0.885
107 131
1.33 (0.06–188.28) 2.24 (0.08–197.10)
0.578
0.08 (0–25.13) 0.33 (0–24.88)
0.722
91 147
4.34 (0.08–76.05) 1.33 (0.06–197.10)
0.059
0.43 (0.01–24.75) 0.25 (0–25.13)
0.879
19 101 118
2.72 (0.47–77.24) 4.48 (0.08–188.28) 1.27 (0.06–197.10)
0.361
0.53 (0–22.23) 0.79 (0–25.13) 0.25 (0–24.88)
0.327
93 145
2.72 (0.08–188.28) 1.5 (0.06–197.10)
0.957
0.32 (0–24.88) 0.08 (0–25.13)
0.059
111 127
4.18 (0.11–177.49) 1.29 (0.06–197.10)
0.321
0.25 (0–25.13) 0.33 (0–24.85)
0.712
108 130
1.22 (0.08–76.05) 4.48 (0.06–197.10)
0.041
0.33 (0–24.88) 0.18 (0–25.13)
0.993
Stage III IV Chemotherapy Platinum-based with TAX Platinum-based without TAX Response to chemotherapy Responder (CR+PR) Non-responder (SD+PD)
TAX paclitaxel, CR complete response, PR partial response, SD stable disease, PD progression disease
Tumor Biol. Table 3 Association of protein expression levels of stathmin and β-IIItubulin with clinical features
Characteristic
All patients
Stathmin expression
(n=238)
Low (n)
High (n)
126 112
56 54
70 58
162 76
79 31
107 131
P
β-III-tubulin expression
P
Low (n)
High (n)
0.560
25 24
101 88
0.762
83 45
0.250
36 13
126 63
0.363
46 64
61 67
0.367
21 28
86 103
0.740
91 147
40 70
51 77
0.582
18 31
73 116
0.808
19 101 118
12 54 46
7 47 72
0.035
5 20 24
14 81 94
93 145
45 67
48 78
0.742
19 30
74 115
0.961
127 111
66 46
61 65
0.105
27 22
100 89
0.784
108 130
60 52
48 78
0.017
24 25
84 105
0.570
Age (range), year ≤60 >60 Gender Male Female Smoking status Never Current or ever Pathological type Squamous cell carcinoma Adenocarcinoma Tumor differentiation Well Moderate Poor Stage III IV Chemotherapy Platinum-based with TAX Platinum-based without TAX Response to chemotherapy Responder (CR+PR) Non-responder (SD+PD)
TAX paclitaxel, CR complete response, PR partial response, SD stable disease, PD progression disease
was tended to be poor differentiation (P=0.035) and poor response to chemotherapy (P=0.017). However, no significant relationship was found between β-III-tubulin expression and any of the listed clinical features (P>0.05).
Correlation of stathmin and β-III-tubulin mRNA expression with survivals The OS and PFS using the Kaplan–Meier analysis revealed that the prognosis of NSCLC patients with high stathmin mRNA expression was significantly poorer than those with low stathmin expression (for OS: log-rank P =0.012; for PFS: log-rank P=0.039). Moreover, stratified analysis by treatment indicated that the associations between stathmin expression level and OS and PFS were substantial in patients who were treated by both platinum and TAX (for OS: log-rank P = 0.010; for PFS: log-rank P = 0.031, respectively). Similarity in survival differences were not found for patients who were treated by platinum without TAX (for OS: log-rank P=0.367; for PFS: log-rank P=0.457, respectively) (Figs. 1 and 2).
As for β-III-tubulin, Kaplan–Meier plots showed that β-IIItubulin expression was tended to be associated with PFS but not OS. When compared to low expression, patients with higher expression level of β-III-tubulin was slightly correlated with shorter PFS (log-rank P=0.163). The results stratified by treatment regimens indicated that the slight correlation between β-IIItubulin expression and PFS was significant only among patients who were treated by both platinum and TAX (log-rank P=0.038). Similar PFS difference was not found for patients who received both platinum without TAX (log-rank P=0.859) (Fig. 3). Correlation of stathmin and β-III-tubulin protein expression with survivals The correlation of stathmin and β-III-tubulin protein expression with survivals and response were consistent with the correlation of their mRNA expression with survivals and response. In brief, high stathmin protein expression were significantly associated with shorter OS and PFS than low stathmin expression (for OS: log-rank P=0.014; for PFS: log-rank P= 0.022). Moreover, the associations were still significant only among patients who were treated by both platinum and TAX
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Fig. 1 Kaplan–Meier curves for overall survival according to stathmin expression in patients with NSCLC. a Overall survival according to stathmin mRNA level in patients who were treated by platinum-based chemotherapy without paclitaxel. b Overall survival according to stathmin mRNA level in patients who were treated by platinum-based
chemotherapy combined with paclitaxel. c Overall survival according to stathmin protein level in patients who were treated by platinum-based chemotherapy without paclitaxel. d Overall survival according to stathmin protein level in patients who were treated by platinum-based chemotherapy combined with paclitaxel
(for OS: log-rank P=0.005; for PFS: log-rank P=0.016, respectively). Similar differences of survival were not found for patients who were treated by platinum-based chemotherapy without TAX (for OS: log-rank P=0.626; for PFS: log-rank P=0.422, respectively) (Figs. 1 and 2). Patients with higher β-III-tubulin expression were slightly correlated with shorter PFS (log-rank P=0.078) when compared to lower expression. Stratified analysis showed that the correlation between β-III-tubulin expression and PFS was significant only among patients who were treated by platinumbased chemotherapy without TAX (log-rank P=0.094) (Fig. 3).
high. The limited efficacy of cytotoxic chemotherapy and anticancer drug resistance are major obstacles for the treatment of advanced lung cancer patients [24]. Antimicrotubule agents are widely used chemotherapy drugs for lung cancer treatment [25]. Currently, the relationship between microtubule dynamics and TAX resistance attracted more and more attention; therefore, β-IIItubulin and stathmin which can interact with microtubules become research hotspots. β-III-tubulin has been associated with chemotherapeutic resistance to the microtubulebinding protein; however, the results are inconsistent [13]. Previous studies in endometrial and lung cancer cell lines showed that increased levels of stathmin were resistant to TAX [17, 26]; however, the role of stathmin expression in TAX resistance was detected by few studies in vivo. In our present study, we investigated the relationship between β-III-tubulin, stathmin expression (both in mRNA
Discussion There are significant advances in oncology in recent decades, even though the mortality of lung cancer remains
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Fig. 2 Kaplan–Meier curves for progression-free survival according to stathmin expression in patients with NSCLC. a Progression-free survival according to stathmin mRNA level in patients who were treated by platinum-based chemotherapy without paclitaxel. b Progression-free survival according to stathmin mRNA level in patients who were treated by platinum-based chemotherapy combined with paclitaxel. c
Progression-free survival according to stathmin protein level in patients who were treated by platinum-based chemotherapy without paclitaxel. d Progression-free survival according to stathmin protein level in patients who were treated by platinum-based chemotherapy combined with paclitaxel
and protein levels), and clinical outcome in NSCLC patients who received platinum-based chemotherapy with and without TAX. Our results showed that stathmin expression (both in mRNA and protein levels) was not only associated with PFS and OS but also was related to chemotherapy response. However, βIII-tubulin expression (both in mRNA and protein levels) was only associated with PFS but not OS; besides, β-III-tubulin expression was not correlated to chemotherapy response. Of note, the associations were substantial only in patients who were treated by both platinum and TAX, but not in patients who were treated by platinum without TAX. These findings suggest that the expression level of stathmin (or β-III-tubulin) has an impact on the therapeutic effect of TAX. Stathmin, which is considered as a microtubuledestabilizing protein, can modulate microtubule dynamics
through preventing polymerization of tubulin and boosting destabilization and disassembly of microtubule during cell cycle progression [16]. Hence, elevated expression level of stathmin may play a role in disturbing microtubule stabilization; therefore, the influence of TAX on microtubule polymerization may be affected. Besides, stathmin is implicated in the regulation process of cell proliferation, mobility, metastasis, and differentiation, which may also have an impact on tumor progression [16, 19]. Our results suggest that stathmin mRNA or protein expression level can be considered as a strong predictive marker of NSCLC survival after platinum and TAX chemotherapy. There are several possible mechanisms to elucidate our findings; TAX resistance is considered as one of the probabilities. It was demonstrated by a previous study that targeting stathmin and Bcl-2 expression with RNA interference (RNAi) significantly downregulates RNA and protein
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Fig. 3 Kaplan–Meier curves for progression-free survival according to β-III-tubulin expression in patients with NSCLC. a Progression-free survival according to β-III-tubulin mRNA level in patients who were treated by platinum-based chemotherapy without paclitaxel. b Progression-free survival according to β-III-tubulin mRNA level in patients who were treated by platinum-based chemotherapy combined
with paclitaxel. c Progression-free survival according to β-III-tubulin protein level in patients who were treated by platinum-based chemotherapy without paclitaxel. d Progression-free survival according to β-III-tubulin protein level patients who were treated by platinum-based chemotherapy combined with paclitaxel
expression levels of stathmin and Bcl-2 and sensitizes lung cancer cells to TAX [26]. In addition, in breast cancer cell lines, higher expression level of stathmin diminished microtubule polymerization, leading to cell cycle arrest at G2 phase, thus evidently decreased the binding of TAX to microtubules [27]; on the contrary, stathmin suppression by RNAi promoted microtubule polymerization and facilitated cell cycle progression from G2 to M phase; therefore, cell sensitivity to TAX was elevated [28]. Besides, two TAX-resistant ovarian cancer cell lines exhibit higher expression level of stathmin [29]. Furthermore, in prostate cancer cell lines, stathmin inhibition and TAX exposure had a synergistic activity on proliferation and angiogenesis [30]. In summary, all the above findings indicate that stathmin may interfere with tumor response to TAX treatment. The association between stathmin expression (both in mRNA and protein levels) and NSCLC
prognosis in our present study becomes another evidence that stathmin may be implicated in lung cancer resistance to TAX treatment. In our current study, β-III-tubulin expression was associated with PFS but was not related to chemotherapy response. As for this result, we need to give comprehensive explanations. To our knowledge, there is a supposition that the prognosis of patients will be better if they initially respond well to chemotherapy; therefore, the response to treatment has become an early predictive marker [31]. However, patients who have comparable response to initial treatment can result in different clinical outcomes. The associations between treatment response and survival are different in different studies because of inconsistent treatment protocols and evaluation criterion of response. Of note, in our study, response to treatment was assessed after two treatment cycles. There is a possibility that
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the treatment response was evaluated too soon; thus far, the correction between response and prognosis was not observed. In addition, although the results showed that β-III-tubulin was related to PFS but not OS, the complication of clinical and pathological differentiation among lung cancer patients may also have resulted in the inability to investigate the relations between β-III-tubulin and treatment response. Interestingly, the results also showed that the expression of β-III-tubulin (both in mRNA and protein levels) tended to be associated with NSCLC survival, but the relation was only observed in progression-free survival among patients who were treated by platinum-based chemotherapy without TAX. We need to consider the data carefully. Previous studies in vitro have suggested that overexpression of β-III-tubulin can lead to TAX resistance through decreasing microtubule assembly and reducing the ability of TAX to prohibit microtubule dynamics [32]. Results from previous studies also demonstrated associations between higher level of β-III-tubulin and TAX resistance in patients with ovarian cancer [21], breast cancer [33], and NSCLC [34], which are consistent with our results. Although previous studies also showed inconsistent results [13], combined with our results, we considered that platinum-based chemotherapy with TAX regimen may be a little more beneficial for patient with NSCLC, compared with platinum-based chemotherapy without TAX regimen. Further investigations should be carried out on the predictive value of βIII-tubulin expression in TAX resistance, chemotherapy response, and survival of a larger and independent cohort in the future. In summary, although the underlying mechanism remains to be elucidated, the results showed that stathmin expressions (both in mRNA and protein levels) are important predictive markers for NSCLC patients who are treated by platinum-based chemotherapy combined with TAX. Besides, overexpression of stathmin was significantly associated with shorter progression-free survival and overall survival. Our findings demonstrate that stathmin may play a role in interfering the platinum and TAX treatment on NSCLC patients, and expression of stathmin should be investigated in TAX treatment in patients with NSCLC in further studies.
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Acknowledgments This work was partly supported by Postdoctoral Science Foundation of China (No. 2014M552461). We would like to thank all the participants and staff of Shaan’Xi Tumor Hospital for their valuable contributions on collecting specimens and sorting out clinical data. Conflicts of interest None
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