Knockdown of STMN1 enhances osteosarcoma cell chemosensitivity through inhibition of autophagy.

Chemoresistance is a major cause for the poor prognosis of osteosarcoma (OS) patients. However, our understanding of mechanisms underlying chemoresistance in OS are limited. The present study aimed to investigate the effect of stathmin 1 (STMN1) on paclitaxel-induced chemoresistance, as well as the underlying mechanism. Western blot analysis data revealed that the expression level of STMN1 was dramatically increased in OS cell lines (HOS, Saos-2, U-2OS and MG-63), when compared to normal osteoblast hFOB1.19 cells. Furthermore, treatment with paclitaxel led to upregulation of STMN1 in U-2OS cells, accompanied by activation of autophagy, which may attenuate the cytotoxicity of paclitaxel in OS cells. Following knockdown of STMN1 expression, paclitaxel-induced autophagy was significantly reduced, accompanied by increased cytotoxicity of paclitaxel to U-2OS cells. In addition, blockade of mammalian target of rapamycin signaling attenuated the inhibitory effect of STMN1 knockdown on autophagy in OS cells. In conclusion, the present study demonstrated that knockdown of STMN1 enhances osteosarcoma cell chemosensitivity to paclitaxel through inhibition of autophagy. Therefore, STMN1 may be a potential target for the treatment of chemoresistant OS.

Introduction

As the most common mesenchymal sarcoma in bone, osteosarcoma (OS) mainly arises from the metaphysis of the long bones (1). Although great efforts have been made in OS diagnosis and therapy, chemoresistance remains a major problem that causes low 5-year survival rates (1). Furthermore, the understanding of the mechanism underlying chemoresistance is limited. It has been demonstrated that various oncogenes and tumor suppressors are involved in chemoresistance (2–4). Thus, investigation into the molecular targets involved in OS cell chemoresistance is urgently required.

Autophagy, an evolutionarily conserved function, is a cellular self-catabolic degradation process, responsible for the lysosomal degradation of long-lived proteins as well as aged or damaged organelles (5). The amino acids and fatty acids generated during autophagy may be reused and thus autophagy may be beneficial for sustainable cell survival (6). Accumulating evidence has been reported that autophagy is rapidly activated when cancer cells are treated with certain chemotherapy drugs (7,8), and functions as a protective mechanism to promote cancer cell survival (5). Therefore, autophagy frequently contributes to chemoresistance, and inhibition of autophagy has been observed to promote chemotherapy efficacy (9).

Stathmin 1 (STMN1), a cytosolic phosphoprotein, has been reported to be abundantly expressed in various cell types (10,11). Furthermore, STMN1 is able to regulate microtubule dynamics by promoting depolymerization of microtubules and/or preventing polymerization of tubulin heterodimers (12). Therefore, anti-STMN1 therapy has been reported to improve sensitivity to antimicrotubule drugs in esophageal squamous cell carcinoma (12). Zhang et al (13) reported that the expression of STMN1 was significantly increased in two human OS cell lines (SOSP-9607 and SOSP-9901) and 45 OS tissue specimens, compared with normal tissues. It was also demonstrated that knockdown of STMN1 inhibited OS cell proliferation and cell cycle progression, while inducing apoptosis (13), suggesting that STMN1 may act as an oncogene in OS. Phadke et al (14) evaluated the in vivo safety and antitumor efficacy of bifunctional small hairpin RNAs specific for STMN1. The results of this previous study confirmed the systemic safety of the therapeutic dose, and thus supported the early-phase assessments of clinical safety and preliminary efficacy (14). However, to the best of our knowledge, there have been no reports of the role of STMN1 in the regulation of chemosensitivity in OS.

The present study aimed to investigate the effect of STMN1 on paclitaxel-induced chemoresistance, as well as the underlying mechanism of action.

Materials and methods

Cell culture

OS cell lines HOS, Saos-2, U-2OS and MG-63, and normal osteoblast cell line hFOB1.19, were obtained from American Type Culture Collection (Manassas, VA, USA). All cell lines were cultured in Dulbecco's modified Eagle's medium (Thermo Fisher Scientific, Inc., Waltham, MA, USA) with 10% fetal bovine serum (Thermo Fisher Scientific, Inc.) at 37°C in a humidified incubator containing 5% CO2.

Cell transfection or treatment

The recombinant lentivirus anti-STMN1 (GeneChem Co., Ltd., Shanghai, China), as well as the control anti-NC (negative control; GeneChem Co., Ltd.) were transfected into U-2OS cells by using Lipofectamine 2000 (Thermo Fisher Scientific, Inc.). Stable transfected cells were constructed using G418 (Thermo Fisher Scientific, Inc.) selection. Cells in each group were treated with 3 µM paclitaxel (Sigma-Aldrich; KGaA, Darmstadt, Germany) for 3 h at 37°C. The anti-STMN1 and anti-NC U-2OS cells were treated with 5 µM LY29054 (Selleck Chemicals, Houston, TX, USA).

Reverse transcription-quantitative polymerase chain reaction (qPCR)

Total RNA was prepared using TRIzol reagent (Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. For the analysis of mRNA expression, RevertAid™ H Minus First Strand cDNA Synthesis kit (Thermo Fisher Scientific, Inc.) was used to reverse transcribe RNA into cDNA, and qPCR was subsequently performed using the Power SYBR Green PCR Master mix (Bio-Rad Laboratories, Inc., Hercules, CA, USA) on an ABI 7500 thermocycler (Thermo Fisher Scientific, Inc.). The cycling conditions were as follows: 95°C for 5 min, followed b7 40 cycles of 95°C for 10 sec and 60°C for 30 sec. The primer sequences for STMN1 were as follows: Sense, 5′-TCAGCCCTCGGTCAAAAGAAT-3′ and antisense, 5′-TTCTCGTGCTCTCGTTTCTCA-3′. The primer sequences for GAPDH were as follows: Sense, 5′-GGAGCGAGATCCCTCCAAAAT-3′ and antisense, 5′-GGCTGTTGTCATACTTCTCATGG-3′. GAPDH was used as an endogenous control. The relative expression was analyzed by the 2−ΔΔCq method (15).

Western blot analysis

Protein was extracted from cells using radioimmunoprecipitation assay buffer (Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Subsequently, protein was quantified using the Pierce Protein Assay kit (Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol Proteins (50 µg) were separated by 12% SDS-PAGE, transferred to polyvinylidene difluoride membranes and probed with primary antibodies: Rabbit anti-STMN1 antibody (1:100; ab52630; Abcam, Cambridge, MA, USA), rabbit anti-LC3B antibody (1:50; ab48394; Abcam), rabbit anti-Beclin1 antibody (1:100; ab62557; Abcam), rabbit anti-mammalian target of rapamycin (mTOR) antibody (1:100; ab2732; Abcam), rabbit anti-phosphorylated (p)-mTOR (1:100; ab109268; Abcam) or rabbit anti-GAPDH antibody (1:50; ab9485; Abcam) at 4°C overnight. Membranes were subsequently incubated with mouse anti-rabbit secondary antibody (1:10,000; ab99697; Abcam) at room temperature for 40 min. The protein bands were visualized by the Amersham enhanced chemiluminescence system (RPN998; GE Healthcare Life Sciences, Chalfont, UK). Data was analyzed by densitometry using Image-Pro plus software version 6.0 (Media Cybernetics, Inc., Rockville, MD, USA) and were normalized to GAPDH expression.

Cell survival assay

U-2OS cells in each group were seeded into 10 mm dishes, and incubated for 14 days. Subsequently, cells were fixed in methanol for 15 min, stained with Giemsa (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) for 10 min and dried in air. The number of colonies was counted under a microscope (CX23; Olympus Corporation, Tokyo, Japan).

Statistical analysis

Data in the figures are expressed as the mean ± standard deviation. Analysis of data was performed using SPSS version 17 (SPSS, Inc., Chicago, IL, USA). Student's t-test or one-way analysis of variance were used depending on the experimental conditions. P<0.05 was considered to indicate a statistically significant difference.

Results

Treatment with paclitaxel leads to activation of autophagy, as well as upregulation of STMN1 in OS cells

The present study performed western blot analysis to examine the mRNA and protein levels of STMN1 in OS cell lines. As presented in Fig. 1A and B, STMN1 was significantly upregulated in OS cell lines (Saos-2, U-2OS, HOS and MG-63), when compared to normal osteoblast hFOB1.19 cells. As U-2OS cells demonstrated the highest level of STMN1, this cell line was used in the subsequent experiments. U-2OS cells were treated with paclitaxel for 3 h, and the cell survival and autophagy were examined. It was observed that treatment with paclitaxel inhibited cell survival (Fig. 2A). However, it also led to activation of autophagy, demonstrated by the upregulated protein levels of Beclin1 and LC3II, as well as the increased radio of LC3II to LC3I (Fig. 2B and C). In addition, it was also observed that STMN1 was significantly upregulated in U-2OS cells following exposure to paclitaxel (Fig. 2D), suggesting that STMN1 may be associated with paclitaxel-induced autophagy in U-2OS cells.

Figure 1.

(A) Reverse transcription-quantitative polymerase chain reaction and (B) western blot analysis were performed to determine the mRNA and protein levels of STMN1 in four osteosarcoma cell lines (HOS, Saos-2, U-2OS and MG-63, as well as normal osteoblast cell line hFOB1.19). **P<0.01 vs. hFOB1.19. STMN1, stathmin 1.

Figure 2.

(A) MTT assay was performed to examine the survival of U-2OS cells treated with paclitaxel for 3 h. (B) Western blot analysis was conducted to determine the protein levels of autophagy-associated proteins. (C) The ratio of LC3II to LC3I was determined. (D) Western blot analysis was conducted to determine the protein levels of STMN1. Non-treated U-2OS cells were used as a control. **P<0.01 vs. control. LC, light chain; STMN1, stathmin 1; OD, optical density.

Knockdown of STMN1 expression sensitizes U-2OS cells to paclitaxel

To confirm that STMN1 was involved in paclitaxel-induced autophagy in U-2OS cells, U-2OS cells with stably decreased expression of STMN1 were induced (Fig. 3A and B). The present study also investigated the role of STMN1 in the sensitivity of OS to paclitaxel. Cells in each group were treated with paclitaxel for 3 h, and the cell survival was examined. As shown in Fig. 3C, the cell survival in the anti-STMN1 group was significantly decreased, when compared with that in the anti-NC group and the control group. Furthermore, there was no significant change between the anti-NC group and control group. These data suggest that knockdown of STMN1 expression enhanced the sensitivity of U-2OS cells to paclitaxel.

Figure 3.

(A) Reverse transcription-quantitative polymerase chain reaction and (B) western blot analysis were conducted to determine the mRNA and protein levels of STMN1 in U-2OS cells transfected with lentivirus anti-STMN1 or the control anti-NC. (C) MTT assay was performed to examine the cell survival in each group. Non-transfected U-2OS cells were used as a control. **P<0.01 vs. control. STMN1, stathmin 1; NC, negative control; OD, optical density.

Knockdown of STMN1 expression inhibits paclitaxel-induced autophagy in OS cells

Western bolt analysis was conducted to determine the levels of autophagy-associated proteins in each group. It was observed that the protein levels of Beclin1 and LC3II (Fig. 4A), as well as the ratio of LC3II to LC3I (Fig. 4B), were reduced in the anti-STMN1 group, when compared with those in the anti-NC group and the control group. These data suggest that knockdown of STMN1 inhibited paclitaxel-induced autophagy in OS cells.

Figure 4.

(A) Western blot analysis was conducted to determine the protein levels of Beclin1 in U-2OS cells transfected with lentivirus anti-STMN1 or the control anti-NC. (B) Western blot analysis was conducted to determine the protein levels of LC3II and LC3I, and the ratio of LC3II to LC3I was determined. Non-transfected U-2OS cells were used as a control. **P<0.01 vs. control. STMN1, stathmin 1; NC, negative control; LC, light chain.

Blockade of mTOR signaling attenuates the inhibitory effect of STMN1 knockdown on autophagy in OS cells treated with paclitaxel

The molecular mechanisms underlying the role of STMN1 in autophagy were investigated. The activity of mTOR signaling was evaluated by using western blot analysis. It was observed that knockdown of STMN1 expression increased the levels of p-mTOR in OS cells treated with paclitaxel (Fig. 5A), suggesting that the activation of mTOR signaling may be responsible for the inhibitory effect of STMN1 knockdown on paclitaxel-induced autophagy in OS cells. To additionally confirm this hypothesis, LY294002 was applied to inhibit the activity of mTOR signaling. STMN1 silenced U-2OS cells were treated with or without LY294002, followed by paclitaxel, and the levels of mTOR signaling- and autophagy-associated proteins were examined. As presented in Fig. 5B, STMN1 silenced U-2OS cells treated with LY294002 and paclitaxel demonstrated a reduced p-mTOR level, when compared with that in STMN1 silenced U-2OS cells treated with paclitaxel alone, indicating that the activity of mTOR signaling was reduced. Furthermore, STMN1 silenced U-2OS cells treated with LY294002 and paclitaxel demonstrated increased levels of Beclin1 and LC3II, as well as the ratio of LC3II/LC3I, when compared with those in STMN1 silenced U-2OS cells treated with paclitaxel alone, indicating that the autophagy level was increased (Fig. 5C and D). Therefore, inhibition of mTOR activity attenuated the inhibitory effect of STMN1 knockdown on paclitaxel-induced autophagy in U-2OS cells, indicating that STMN1 participates in paclitaxel-induced autophagy through mediation of mTOR signaling.

Figure 5.

(A) Western blot analysis was conducted to determine the protein levels of p-mTOR and total mTOR in U-2OS cells transfected with lentivirus anti-STMN1 or the control anti-NC. Non-transfected U-2OS cells were used as a control. **P<0.01 vs. control. (B) Western blot analysis was conducted to determine the protein levels of p-mTOR and total mTOR in STMN1 silencing U-2OS cells treated with or without LY294002, followed by paclitaxel treatment. (C) Western blot analysis was conducted to determine the protein levels of Beclin1 in each group. (D) Western blot analysis was conducted to determine the protein levels of LC3II and LC3I, and the ratio of LC3II to LC3I was determined. **P<0.01 vs. anti-STMN1. p, phosphorylated; mTOR, mammalian target of rapamycin; STMN1, stathmin 1; NC, negative control; LC, light chain.

Discussion

Investigation of the molecular mechanisms associated with chemoresistance is important for chemotherapeutic treatment of OS. In the present study, it was observed that STMN1 was significantly upregulated in OS cell lines compared with normal osteoblast hFOB1.19 cells. Treatment with paclitaxel enhanced the expression of STMN1 in U-2OS cells, accompanied by activation of autophagy. Knockdown of STMN1 expression suppressed paclitaxel-induced autophagy and increased the cytotoxicity of paclitaxel to U-2OS cells. Molecular mechanism investigation revealed that knockdown of STMN1 expression activated mTOR signaling in OS cells, while blockade of mTOR signaling attenuated the inhibitory effect of STMN1 knockdown on autophagy in OS cells. Therefore, the present study demonstrated that knockdown of STMN1 enhances osteosarcoma cell chemosensitivity through inhibition of autophagy, and mTOR signaling is involved in this process.

STMN1 has been reported to be an oncogene, which is expressed at high levels in a wide variety of human malignancies, and serves important roles in maintenance of malignant phenotypes (16). For instance, Hemdan et al (17) reported that the expression of STMN1 was significantly increased in bladder cancer tissues, was correlated with reduced disease-specific survival and 70% of patients were STMN1-positive in primary tumor and matched metastases groups, suggesting that STMN1 expression has prognostic significance, and may be a potential treatment target in bladder cancer. Furthermore, STMN1 is also upregulated in esophageal carcinoma, and increased expression levels of STMN1 are associated with low 5-year survival rates of patients with esophageal carcinoma (18). In addition, STMN1 was observed to be upregulated in OS tissues compared with normal tissues (13). The present study also observed that expression levels of STMN1 were significantly increased in four OS cell lines (HOS, Saos-2, U-2OS and MG-63) compared with normal osteoblast hFOB1.19 cells. Therefore, upregulation of STMN1 may be also involved in the development of OS. However, to the best of our knowledge, the exact role of STMN1 in OS cell chemoresistance has not previously been studied. The present study observed that treatment with paclitaxel in OS cells led to a significant upregulation of STMN1 expression, as well as autophagy, which has been reported to be associated with chemoresistance in multiple types of human cancers including OS. Guo et al also reported that paclitaxel induced apoptosis accompanied by protective autophagy in OS cells via the hypoxia-inducible factor-1α signaling pathway (19).

Furthermore, inhibition of autophagy has been reported to have negative effects on OS. Knockdown of autophagy-associated Beclin1 decreased cell proliferation, invasion and metastasis, and had a positive effect on chemotherapy-induced cytotoxicity in OS cells (20). In the present study, it was observed that knockdown of STMN1 led to a significant decrease in the expression of Beclin1, as well as the ratio of LC3II to LC3I, and paclitaxel-induced cytotoxicity in OS cells was enhanced. Wu et al (21) reported that inhibition of Beclin1 enhanced the chemotherapeutic sensitivity of osteosarcoma cells to cisplatin.

The mTOR signaling pathway has been demonstrated to serve a suppressive role in autophagy, and inhibition of mTOR signaling causes upregulation of autophagy (22,23). Chang et al (24) reported that dual phosphoinositide 3-kinase/mTOR inhibitor NVP-BEZ235-induced apoptosis of hepatocellular carcinoma cell lines was enhanced by inhibition of autophagy. In the present study, it was observed that knockdown of STMN1 led to upregulation of mTOR signaling, which additionally inhibited autophagy. Therefore, LY294002-induced blockade of mTOR signaling attenuated the inhibitory effect of STMN1 knockdown on autophagy in OS cells treated with paclitaxel.

In addition, STMN1 has been demonstrated to be a direct target of microRNA (miR)-101, and is involved in miR-101-mediated inhibition of autophagy in several types of human cancer (25–27). Frankel et al (28) reported that miR-101 was able to inhibit etoposide- and rapamycin-induced autophagy, while overexpression of STMN1 partially rescued cells from miR-101-mediated inhibition of autophagy. Xu et al (29) reported that miR-101 inhibited autophagy and enhanced cisplatin-induced apoptosis in hepatocellular carcinoma cells, partly at least, via direct inhibition of STMN1 expression. In addition, STMN1 was observed to be associated with radioresistance in human cancer. Sun et al (30) reported that miR-101 sensitizes human nasopharyngeal carcinoma cells to radiation by targeting STMN1.

In conclusion, the present study demonstrated that knockdown of STMN1 enhances osteosarcoma cell chemosensitivity to paclitaxel via inhibition of autophagy. Therefore, STMN1 may be a potential target for the treatment of chemoresistant OS.