Oncogene (2016), 1–11 © 2016 Macmillan Publishers Limited All rights reserved 0950-9232/16 www.nature.com/onc

ORIGINAL ARTICLE

Microtubule-associated protein 4 is an important regulator of cell invasion/migration and a potential therapeutic target in esophageal squamous cell carcinoma Y-Y Jiang1, L Shang1, Z-Z Shi1, T-T Zhang1, S Ma1, C-C Lu2, Y Zhang1, J-J Hao1, C Shi1, F Shi1, X Xu1, Y Cai1, X-M Jia2, Q–M Zhan1 and M-R Wang1 Cell invasion and migration significantly contribute to tumor metastasis. Microtubule-associated protein 4 (MAP4) protein is one member of microtubule-associate proteins family. It is responsible for stabilization of microtubules by modulation of microtubule dynamics. However, there is little information about the involvement of MAP4 in human cancer. Here we show that MAP4 serves as a regulator of invasion and migration in esophageal squamous cancer cells. By activating the ERK-c-Jun-vascular endothelial growth factor A signaling pathway, MAP4 promotes cell invasion and migration in vitro, tumor growth and metastasis in mouse models. Immunohistochemical staining of operative tissues indicated that MAP4 expression was associated with tumor stage, lymph node metastasis and shorter survival of the patients with esophageal squamous cell carcinoma (ESCC). Multivariate Cox regression analysis showed that MAP4 is an independent prognostic indicator. In the serial sections of ESCC tissues, there was a positive correlation between MAP4 and vascular endothelial growth factor A expression. Notably, an intratumoral injection of MAP4-small interfering RNA (siRNA) remarkably inhibited the growth of the tumors that formed by the MAP4-expressing ESCC cells in nude mice, and a combination of MAP4-siRNA and Bevacizumab significantly enhanced the inhibition effect. Our data suggest that MAP4 is probably a useful prognostic biomarker and a potential therapeutic target for the disease. Oncogene advance online publication, 15 February 2016; doi:10.1038/onc.2016.17

INTRODUCTION Esophageal cancer is the sixth leading cause of cancer related death in the world. Esophageal squamous cell carcinoma (ESCC) is the predominant type of esophageal cancer occurring in the Chinese population.1 Tumor invasion and migration are crucial steps in the metastatic process. A multitude of molecular changes could enable tumor cells to acquire highly motile properties. Major challenges in treating ESCC are to identify effective therapeutic targets that can inhibit cancer metastasis and reduce the mortality. We previously revealed that cortactin (CTTN) gene is an oncogene in promoting the metastasis of ESCC.2 In the further investigation, we recently found that microtubule-associated protein 4 (MAP4) is in the CTTN-complex by GST-pull down and liquid chromatography–mass spectrometry (LC-MS)/MS analyses. MAP4 gene is located on chromosome 3p21, it is one member of MAPs family with an N-terminal projection domain and a C-terminal microtubule-binding domain, and responsible for stabilization of microtubules by modulation of microtubule dynamics through interaction with Cyclin B, p38 or Septins.3–6 It has been reported that MAP4 affects cell cycle progression7 and apoptosis.8 Up to date, there is little information about the involvement of MAP4 in human cancer. A recent study showed that MAP4 promotes cell invasion in bladder cancer,9 but the underlying mechanisms remain unclear. We here report that MAP4 protein levels were upregulated in ESCC tissues compared with the normal esophageal epithelia. 1

MAP4 and vascular endothelial growth factor A (VEGFA) expression was positively correlated in clinical specimens. Mechanistically, MAP4 promoted cell invasion and migration via MAP4-ERK-Jun-VEGF signaling, in which VEGFA might exert function independent of angiogenesis. RESULTS MAP4 is an independent prognostic factor for ESCC Our previous studies have demonstrated an essential role of CTTN in ESCC, we hypothesized that other proteins interacted with CTTN may also be important in this disease. We therefore identified CTTN-associated proteins by GST-pull down and LCMS/MS. Of the proteins identified, MAP4 was selected for further investigation as there is little information concerning the role of this protein in cancer (Supplementary Figure S1 and Supplementary Table S1). We first performed a separate knockdown of MAP4 and CTTN with the technique of RNA interference and found that there was no obvious regulating relationship between CTTN and MAP4 (Supplementary Figure S2). Subsequently, we detected MAP4 expression in operative ESCC specimens by using immunohistochemistry technique combined with tissue microarray. In the 364 ESCCs, high MAP4 expression was observed in 228 (62.6%) tumors (Figure 1a). The high expression of MAP4 was significantly associated with depth of invasion (pT; P = 0.00124) and lymph node metastasis (P = 0.00026; Table 1).

State Key Laboratory of Molecular Oncology, Cancer Institute/Hospital, Peking Union Medical College and Chinese Academy of Medical Sciences, Beijing, China and Department of Histology and Embryology, Anhui Medical University, Hefei, China. Correspondence: Professor M-R Wang, State Key Laboratory of Molecular Oncology, Cancer Institute/Hospital, Peking Union Medical College and Chinese Academy of Medical Sciences, Panjiayuan south 17#, Beijing 100021, China. E-mail: [email protected] Received 12 June 2015; revised 6 December 2015; accepted 11 December 2015 2

MAP4 is a potential therapeutic target for esophageal cancer Y-Y Jiang et al

2 Kaplan–Meier analysis of 88 cases with the follow-up information showed that patients with high MAP4 expression had poor overall survival as compared with those with weak or no MAP4 staining (P = 0.026, Figure 1b). Multivariate Cox regression analysis indicated that sex, lymph node metastasis and MAP4 expression were independent prognostic factors (Table 2).

Figure 1. High expression of MAP4 and correlation with overall survival in ESCC patients. (a) Representative immunohistochemical photos of MAP4 expression in tumor samples and the corresponding normal epithelia. (b) Kaplan–Meier curve showing a correlation between patients with MAP4-negative tumors and higher survival rates.

Table 1. Relationship between protein overexpression and clinicopathologic parameters in tissues Parameter

MAP4 high expression

P-value

Sex Female Male

63.4% (161/254) 60.9% (67/110)

0.65483

Age at diagnosis, y ⩽ 60 460

63.8% (120/188) 61.4% (108/176)

0.62807

Grade G1 G2 G3

67.0% (67/100) 64.5% (136/211) 48.0% (24/50)

0.0580

68.1% (173/254) 50.0% (55/110)

0.00124

pN N0 N1

54.8% (115/210) 73.4% (113/154)

0.00026

AJCC7 stage I/IIA IIB/III

58.9% (53/90) 63.7% (170/267)

0.41781

pT T1/T2 T3/T4

Abbreviations: AJCC7, American Joint Committee on Cancer; pN, lymph node metastases; pT, pathologic T stage.

Oncogene (2016) 1 – 11

MAP4 is involved in invasion and migration of esophageal cancer cells To investigate the implication of MAP4 in esophageal cancer, we determined the messenger RNA and protein level of MAP4 in ESCC cell lines and found that the highest expression of MAP4 was in Eca109 and KYSE150, and the lowest in KYSE180 (Figure 2a and Supplementary Figure S3). MAP4 small interfering RNA (siRNA)-1 and siRNA-2 were used for transiently downregulating MAP4 expression. Forty-eight hours after transfection, both siRNA-1 and siRNA-2 could effectively suppress the expression of MAP4 in Eca109 and KYSE150 cells (Figure 2b). In vitro analysis showed that knockdown of MAP4 had no affect on cell cycle (Supplementary Figure S4), but significantly inhibit cell invasion and migration compared with non-silencing and parental cells detected by wound-healing, haptotactic cell migration and matrigel chemoinvasion assays (Figures 2c and d). When MAP4 expression was restored by transfecting the GV230-MAP4-expression vector, in which the insert sequence of MAP4 corresponding to the siRNA was synonymously mutant, the inhibition impact of MAP4 knockdown on cell invasion and migration could be rescued (Figures 2b and d). Accordingly, transfection of GV230-MAP4expression vector significantly promoted invasion and migration of KYSE180 cells, which had the lowest basal expression of MAP4 and weak abilities of invasion and migration in the cell lines examined (Figure 2e). To confirm that the role of MAP4 promoting the invasion and migration ESCC cells was not influenced by cell proliferation, we conducted a CCK-8 assay in Eca109 and KYSE150 cells. In the first 3 days after cell seeding, there was no difference in proliferation rates between the experimental and control groups (Supplementary Figure S5). These data demonstrated that knockdown of MAP4 drastically suppressed the invasion and migration of Eca109 and KYSE150 cells. VEGFA acts as a downstream effector in MAP4-induced invasion and migration To investigate what are the downstream effectors of MAP4 in esophageal cancer cells, we examined the proteins that have been reported to influence the invasion and migration of malignant cells. Real-time PCR and western blot analysis showed that VEGFA were significantly reduced in MAP4-siRNA transfected Eca109 and KYSE150 cells and also in their culture supernatants. In contrast, knockdown of VEGFA did not affect MAP4 expression (Figure 3a, Supplementary Figure S6). We subsequently detected the levels of VEGFR1 and VEGFR2 expression in Eca109 and KYSE150 cells. The results indicated that VEGFR1 expression was much higher than VEGFR2 (Supplementary Figure S7). As expected, silencing VEGFA and VEGFR1 expression also markedly inhibited the invasion and migration of Eca109 and KYSE150 cells (Figures 3b–d). We analyzed the protein expression of VEGFR1 and VEGFA in ESCC cell lines, and found that VEGFR1 and VEGFA were at a high level in Eca109 and KYSE150 but low expression in KYSE180 (Figure 3e). The invasion and migration abilities of KYSE180 cells were substantially enhanced by adding recombinant human VEGF121 and VEGF165 (hVEGF121 and hVEGF165) (Supplementary Figure S8A) or transfecting with pCS2vegf121 and pCS2vegf165 (Supplementary Figures S8B and C). When we restored the expressions of VEGFA in MAP4-silenced Eca109 and KYSE150, the invasion and migration abilities of the cells were also rescued (Figures 4a and b, Supplementary Figure S9A). © 2016 Macmillan Publishers Limited

MAP4 is a potential therapeutic target for esophageal cancer Y-Y Jiang et al

3 Table 2.

Multivariate Cox regression analysis for survival time of patients with ESCC

Variable

Sex Age pT pN Grade Stage (AJCC7) MAP4

Univariate analysis

Multivariate cox analysis

HR

95% CI

P-value

HR

95% CI

P-value

3.240 0.752 1.063 2.712 1.441 1.772 2.065

1.273–8.249 0.411–1.374 0.583–1.936 1.476–4.982 0.898–2.310 0.882–3.822 1.076–3.965

0.01366 0.35369 0.83844 0.00130 0.13086 0.14442 0.02929

2.981

1.166–7.618

0.02254

2.465

1.337–4.546

0.00386

2.036

1.056–3.923

0.03370

Abbreviations: AJCC7, American Joint Committee on Cancer; CI, confidence interval; MAP4, microtubule-associated protein 4; pN, lymph node metastases; pT, pathologic T stage.

To explore the clinical implication of the above findings, we investigated the relationship between MAP4 and VEGFA expression in 353 ESCC tissues. We observed that 225 (63.7%) tumors had more intense staining of MAP4, and 179 (50.7%) with high expressions of VEGFA. Both MAP4 and VEGFA proteins were located in the cytoplasm of tumor cells. Statistical analysis showed a significantly positive correlation between MAP4 and VEGFA expressions in the ESCC tissues tested (P = 0.0000037; Figure 3f and Table 3). On the basis of the above observation that the VEGFA level in culture supernatant of KYSE150 cells was significantly lower in silenced-MAP4 group than that in the controls, we treated KYSE180 cells by using the conditioned media of KYSE150 with and without MAP4 knockdown. We found that the conditioned media prepared from culture supernatants of KYSE150 cells without MAP4 knockdown significantly enhanced the invasion and migration abilities of KYSE180 when compared with that from MAP4-silenced cells (Supplementary Figure S9B). MAP4 upregulates VEGFA via ERK1/2 signaling To elucidate the molecular mechanism by which MAP4 regulates VEGFA expression and promotes the invasion and migration of ESCC cells, we examined the effect of MAP4-expressing or -silencing on the potential activation of PI3K/AKT,10,11 ERK1/ 2,12,13 p38 (MAPK),14,15 MTOR16,17 and others signaling pathways involved in invasion and migration (Supplementary Figure S10). The results showed that only the phosphorylation levels of ERK1/2 were dramatically decreased in MAP4-silenced Eca109 and KYSE150 compared with the controls (Figure 4c), suggesting that ERK1/2 were the downstream targets of MAP4-mediated signaling that influences the invasion and migration of ESCC cells. Next we examined whether downregulation of ERK1/2 affected VEGFA expression. The results showed that knockdown of ERK1/2 not only inhibited VEGFA expression but also suppressed the invasion and migration of Eca109 and KYSE150 cells (Figure 4c, Supplementary Figure S11). When added hVEGF121 and hVEGF165, the invasion and migration abilities of ERK1/2silenced Eca109 and KYSE150 cells were rescued (Supplementary Figure S11). Co-immunoprecipitation assays showed that MAP4 was co-immunoprecipitated with ERK1/2 (Figure 4d), demonstrating that MAP4 was probably a novel protein-binding partner with ERK1/2 and that MAP4 promoted ESCC cell invasion and migration by activating the ERK-VEGFA pathway. Meanwhile, we observed that knockdown of VEGFA or VEGFR1 decreased the levels of phosphorylated ERK1/2 (Supplementary Figure S12). Collectively, these data suggest that there is a reciprocal regulation between VEGFA/VEGFR1 and ERK1/2, which contributes to the invasion and migration of esophageal cancer cells. © 2016 Macmillan Publishers Limited

MAP4 regulates VEGFA transcription potentially through c-Jun in ESCC cells VEGFA downregulation in MAP4-silenced cells detected by realtime PCR indicated that MAP4 regulates VEGFA transcription in some way. It has been reported that some transcription factors of VEGFA could be regulated by ERK1/2, such as HIF-1a, Statx and c-Jun. We examined the status of these transcription factors in MAP4-silenced cells. The results showed that knockdown of MAP4 or ERK1/2 did not affect the expression of c-Fos, HIF-1a and others, but the phosphorylation level of c-Jun was markedly decreased in MAP4-KD or ERK1/2-KD cells compared with that in the controls. Also, c-Jun-silenced cells presented a great reduction of invasion and migration capability (Figure 4e, Supplementary Figure S13). Online database searching (http://www.cbrc.jp/research/db/ TFSEARCH.html) revealed two potential AP-1-binding elements located -491 and -940 bp upstream of the transcription initiation site in the VEGFA-promoter region. To validate that VEGFA is a transcriptional target of the MAP4-c-Jun pathway, we assessed luciferase activity using the VEGFA reporter construct with the VEGFA-promoter element. The results showed that the luciferase activity derived by the VEGFA promoter was decreased after knockdown of MAP4 or c-Jun expression (Figure 4f). A chromatin immunoprecipitation (ChIP) assay indicated that phosphorylated c-Jun directly bound to the -491 bp region of the human VEGFA promoter (Figure 4g). Taken together, these data suggest that MAP4 regulates VEGFA expression by modulating c-Jun transcriptional activity. MAP4 promotes tumor growth and metastasis in animal models Tumor formation assay in nude mice was performed to investigate the effect of MAP4 on tumor growth in vivo. Eca109 and KYSE150 cells were transduced with sh-MAP4 or sh-scramble lentivirus, and the expression level of MAP4 was confirmed by western blotting (Supplementary Figure S14). The cells with sh-scramble lentivirus were injected subcutaneously into the left hand of the back of BALB/c nude mice, and those with sh-MAP4 in the right hand of the same mice. Both of the sides developed tumors, but the tumors formed from the sh-scramble cells grew obviously faster than those from the sh-MAP4. When the mice were killed at day 18 after injection, tumors formed from sh-MAP4 cells were significantly lighter than those from sh-scramble cells (P o 0.0001; Figures 5a–c). Immunohistochemical staining confirmed the correlation of MAP4 and VEGFA expression in the serial sections of the subcutaneous (s.c.) tumors (Figure 5d, Supplementary Figure S15). We further investigated the influence of MAP4 inhibition on tumor metastasis. The sh-scramble or sh-MAP4 KYSE150 cells were introduced via tail vein into nine NOD/SCID mice, respectively. At day 49 after injection, the mice were killed and examined for the status of lung metastases. In the sh-scramble group, seven of nine Oncogene (2016) 1 – 11

MAP4 is a potential therapeutic target for esophageal cancer Y-Y Jiang et al

4

Figure 2. MAP4 repression decreases cell invasion and migration. (a) Western blot analysis showed that in ESCC cell lines, the highest expression of MAP4 in Eca109 and KYSE150, and the lowest in KYSE180. (b) Western blot analysis showing MAP4 expression in Eca109, KYSE150 and KYSE180 cells after transient transfection. (c) Representative photos of wound-healing assays in parental, non-silencing, MAP4 siRNA Eca109 and KYSE150 cells. (d) Representative photos of haptotactic migration and matrigel chemoinvasion assays in parental of Eca109 and KYSE150, non-silencing, MAP4 siRNA, GV230 and GV230-MAP4 cells. Statistical plots of the haptotactic migration assay and the matrigel chemoinvasion assay. The number of cells transverse the Transwell membranes in MAP4 siRNA and GV230 groups is significantly decreased as compared with parental, non-silencing and GV230-MAP4 groups. (e) Representative photos of haptotactic migration and matrigel chemoinvasion assays in different groups of KYSE180, The number of cells transverse the Transwell membranes in parental and GV230 groups is significantly decreased as compared with GV230-MAP4 groups. The results are expressed as mean ± s.d. of three independent experiments (***P o0.001).

mice developed visually observable lung nodules. But in the sh-MAP4 group, no or little tumor nodules were found either visibly or under the microscope in hematoxylin and eosin sections of mouse lungs (Figure 6a). The average number of lung metastases Oncogene (2016) 1 – 11

per mouse injected with sh-MAP4 cells was significantly lower than that with the sh-scramble (Po0.0001, Figure 6b). Based on that VEGFA has been reported as a secreted protein,18,19 we hypothesized that VEGFA has an important role © 2016 Macmillan Publishers Limited

MAP4 is a potential therapeutic target for esophageal cancer Y-Y Jiang et al

5

Figure 3. MAP4 regulates cell invasion and migration through VEGFA in ESCC cells. (a) Eca109 and KYSE150 cells were transfected with MAP4 siRNA or non-silencing siRNA. Western blotting showing the protein levels of MAP4 and VEGFA, including secreted VEGFA in Eca109 and KYSE150 culture supernatants. (b) Western blot analysis of VEGFA and VEGFR1 expression in non-silencing, VEGFA siRNA and VEGFR1 siRNA cells 48 h after transient transfection. (c) The effect of VEGFA and VEGFR1 expression on cell invasion and migration. Representative photos are shown. Original magnification, × 200. (d) The numbers of VEGFA and VEGFR1-silenced cells that transverse the Transwell chamber are significantly reduced (n = 3, ***P o0.001). (e) Western blot analysis of VEGFA and VEGFR1 expression in ESCC cell lines. (f) MAP4 expression is correlated with VEGFA in human ESCC. Representative photos show consistent expression of MAP4 and VEGFA in tumor tissues.

in the progression of tumor lung metastasis. Therefore, we examined the serum level of VEGFA in the mice, and found that VEGFA content in sh-MAP4 group was obviously lower than that in the sh-scramble (Figure 6c). This observation revealed that MAP4regulated VEGFA exerts function by interacting not only with the VEGFR1 of the tumor cell itself which produces VEGFA but also with that of adjacent tumor cells. In vivo MAP4 silencing and inhibition of VEGFA activity reduces ESCC cell growth We conducted an in vivo experiment with invivofectamine-siRNA complex. We first implanted ESCC cells expressing MAP4 in the back of nude mice to induce tumor formation. When animal tumors reached to about 120 mm3, the mice were randomly divided into the tested group and the control group. The two groups of mice received an intratumoral injection with invivofectamine-MAP4-siRNA complex and invivofectamine-nonsilencing-siRNA complex, respectively. Each mouse was injected with 100 μl of 0.5 mg/ml siRNA complex once every 3 days for three times. At day 10 after the first injection, we observed that © 2016 Macmillan Publishers Limited

the mean tumor volume of the tested group was reduced by 86% as compared with the control group (178.11 mm3 vs 1268.83 mm3; Figures 6d–f). It has been reported that reduction or inhibition of VEGFA expression is associated with reductions in tumor vascularization and angiogenesis with prolonged survival.20 Bevacizumab is a recombinant humanized monoclonal antibody and can inhibit the binding of VEGF to its receptors.21 In view that VEGFA can promote invasion and migration of ESCC cells and that VEGFA content in serum was highly in lung metastases groups in our animal models, we further investigated the inhibitory effect of Bevacizumab on mice tumors. Each nude mouse carrying subcutaneous tumors was injected via tail vein with 100 μl of 5 mg/kg Bevacizumab once every 3 days for three times. At day 10 after the first injection, the mean tumor volume of the control group was 1027.50 mm3, but that of the test group was 265.06 mm3, nearly keeping unchanged during the 10 days (Figure 6d). When combined intratumoral injection of MAP4siRNA with injection of Bevacizumab via tail vein, the tumor volume continuously declined after injection, with a mean Oncogene (2016) 1 – 11

MAP4 is a potential therapeutic target for esophageal cancer Y-Y Jiang et al

6

Figure 4. MAP4 activates ERK1/2 signaling leading to VEGFA activation influence cell invasion and migration ability. (a) Eca109 and KYSE150 cells were transfected with MAP4 siRNA or non-silencing siRNA. At 24 h after transfection, MAP4-silenced cells were transduced with pCS2vegf121 and pCS2vegf165 vector to restore VEGFA expression. pCS2 vector were used as a control. Western blotting shows the levels of MAP4 and VEGFA. (b) The effects of VEGFA expression on cell invasion and migration. Representative photos show that VEGFA expression in MAP4-silenced Eca109 and KYSE150 cells leads to an increase in number of cells that transverse the wells. Original magnification, × 200 (left). The relative number of invasion and migration cells in each chamber (n = 3, ***P o0.001; right). (c) Western blot analysis of MAP4, p-ERK1/2, ERK1/2 and VEGFA after transient transfection of MAP4-siRNA and ERK1/2-siRNA. (d) Western blotting shows an interaction between MAP4 and ERK1/2 in Eca109 and KYSE150 cells. (e) Total and phosphorylated c-Jun protein levels after transient transfection of MAP4 and ERK1/2siRNA. (f) the luciferase activity driven by the VEGFA promoter was significantly decreased after transfection with MAP4-siRNA or c-Jun siRNA. (g) ChIP assay using the chromatin prepared from KYSE150 cells.

Table 3.

The relationship between MAP4 and VEGFA expression in ESCC tissues VEGFA

MAP4

Negative Positive

Total

Total

Negative

Positive

84 90 174

44 135 179

128 225 353

Abbreviations: ESCC, esophageal squamous cell carcinoma; MAP4, microtubule-associated protein 4. Note. P = 0.0000037, Pearson’s Χ2 (two-sided).

Oncogene (2016) 1 – 11

reduction by 94% compared with the control at day 10 (967.5 vs 58.4 mm3; Figures 6d–g). DISCUSSION Extensive local invasion and distant metastasis are the major cause of deaths in patients with ESCC,22 but the molecular mechanisms that drive those process remains elusive. The present study reveals that MAP4 enhances ESCC cells invasion and migration by positively regulating VEGFA via the ERK1/2-c-Jun pathway. MAP4 was initially identified in mouse neuroblastoma cells in 1984.23 It has been shown that MAP4 promotes microtubule assembly and stabilization. However, few observations have © 2016 Macmillan Publishers Limited

MAP4 is a potential therapeutic target for esophageal cancer Y-Y Jiang et al

7

Figure 5. Knockdown of MAP4 expression in Eca109 and KYSE150 suppresses the growth of ESCC xenografts in BALB/c nude mice. (a) Tumor growth curve of mice injected with sh-MAP4 or sh-scramble cells. Data are represented as mean ± s.d. of each group (***Po0.001). (b) At day 18 after injection, larger tumors were formed by sh-scramble cells as compared with the smaller tumors by sh-MAP4 cells in animals. (c) The weights of tumors are presented as mean ± s.d. (***P o0.001, **Po0.01). (d) Immunohistochemical staining of MAP4 and VEGFA in serial sections of subcutaneous tumor tissues. Original magnification, × 100.

correlated MAP4 function with tumor metastasis or poor prognosis in human cancer. In our work, upregulation of MAP4 protein was detected in ESCC tumors compared with adjacent morphologically normal tissues. Furthermore, high MAP4 expression was significantly associated with lymph node metastasis, tumor stage and poor outcome of patients with ESCCs. Multivariate Cox regression analysis revealed MAP4 as an independent prognostic marker, suggesting that MAP4 might have an important part in the progression of ESCC. We characterized key cellular aspects of MAP4 function and found that MAP4 induced cell invasion and migration in vitro, tumor growth and metastasis in vivo. To explore the molecular mechanisms by which MAP4 promoted the malignant phenotypes of ESCC cells, we investigated the potential downstream factors of MAP4 by examining several known pathways and effector molecules associated with cell invasion and migration. We observed that the expression levels of VEGFA and phosphorylated © 2016 Macmillan Publishers Limited

ERK1/2 were dramatically decreased in MAP4-silenced cells compared with the controls. It has been reported that VEGFA production could be regulated by ERK1/2 in some types of cancer cells.24,25 Our co-immunoprecipitation results showed that MAP4 can interact with ERK1/2 in ESCC cells. Also, we observed that knockdown of ERK1/2 inhibited VEGFA expression and suppressed cell invasion and migration. These data suggest a possible mechanism that MAP4 promotes VEGFA expression by interacting with ERK1/2. We further demonstrated that knockdown of MAP4 and ERK1/2 can reduce c-Jun phosphorylation. By Dual Luciferase Reporter and ChIP assay, we showed that phosphorylated c-Jun (not c-Fos) directly bound to the promoter region of human VEGFA and activated the expression of VEGFA. Deng et al.26 reported that both ERK1 and ERK2 could specifically pull down c-Jun protein. Taken together, we hypothesis that MAP4, ERK1/2 and c-Jun co-existed in a complex, and MAP4 promoted ERK1/2 phosphorylation (in an unknown way), which further Oncogene (2016) 1 – 11

MAP4 is a potential therapeutic target for esophageal cancer Y-Y Jiang et al

8

Figure 6. MAP4 promotes lung metastasis of ESCC cells in NOD/SCID mice, and the growth of the tumors formed by MAP4-expressing ESCC cells can be inhibited by an injection of MAP4-siRNA and Bevacizumab. (a) The representative images of lung after Bouin's fixation (top) and hematoxylin and eosin (H&E) staining of tissue sections (bottom). (b) Visible lung metastases were counted and graphed (***P o0.001). (c) The changes of VEGFA’s level detected with a VEGFA ELISA kit in sh-MAP4 and sh-scramble groups. (d) BALB/c nude mice were injected with invivofectamine MAP4-siRNA complex (100 μl of 0.5 mg/ml relative to siRNA) directly into the xenograft tumors or injected with 100 μl of 5 mg/kg Bevacizumab via tail vein every 3 days for three times. The mean tumor volume of test groups is reduced or nearly keeps unchanged as compared with that of control group. When combined an injection of MAP4-siRNA into the xenograft tumors and Bevacizumab via tail vein every 3 days, the tumor volume and size continue to decline. (e) Representative photographs of tumors from each group combined an injection of invivofectamine MAP4-siRNA complex and Bevacizumab, or an injection of invivofectamine non-silencing siRNA complex and 0.9% saline. (f) The weights of tumors are presented as mean ± s.d. (***P o0.001). (g) The representative images of xenograft tumors stained with H&E. Original magnification, × 100.

phosphorylated c-Jun, then the activated c-Jun entered into the nucleus and induced the transcription VEGFA. Further studies are required to investigate how MAP4 positively regulates the phosphorylation of ERK1/2. The role of VEGFA in cancer biology is appearing as an emerging area of importance, despite that most studies on VEGFA have been focused on its functions in angiogenesis and in endothelial cells.27–29 In the present study, higher VEGFA was detected not only in ESCC cells with MAP4 expression, but also in the cultures of such cells and the serum of the mice-bearing cells expressing MAP4 compared with that in silienced-MAP4 cells, their cultures and the serum of the control mice. These data indicate that MAP4 upregulates VEGFA via ERK1/2 signaling, and then VEGFA is secreted into the extracellular milieu and effects biological behavior of tumor cells. Cellular responses to VEGFA are mediated by two high-affinity tyrosine kinase receptors, VEGFR1 (Flt-1) or VEGFR2 Oncogene (2016) 1 – 11

(KDR, Flk1).30–33 Our results indicated that VEGFR1 level is much higher than VEGFR2 in ESCC cells. Silencing VEGFR1 also suppressed cells invasion and migration. Especially, there was a positive correlation between MAP4 and VEGFA expressions in the ESCC tissues. As mentioned above, the activation of ERK1/2 induced the expression of VEGFA, which is responsible for invasion and migration. Meanwhile, we found that silencing VEGFA reduced the phosphorylation level of ERK1/2 in ESCC cells. Some investigations have shown that VEGFA binds to VEGFR2 or VEGFR1 and actives RAS/ERK1/2 signaling pathway.34–36 Taken together with previous findings, we hypothesize that MAP4 exerts function through an autocrine signaling pathway mediated by VEGFA/VEGFR1, which promotes invasion and migration of ESCC cells probably independent of angiogenesis. Furthermore, an addition of hVEGFA or the supernatant from the cultures of cells with high MAP4 expression into the culture media for cells with low MAP4 expression resulted in an enhancement of invasion and © 2016 Macmillan Publishers Limited

MAP4 is a potential therapeutic target for esophageal cancer Y-Y Jiang et al

9 migration abilities of the latter cells. From this evidence we reason that there simultaneously exists a paracrine mechanism involving VEGF release and perception by which the VEGFA secreted by a cancer cell interacts with other adjacent cancer cells and promotes their malignant phenotypes. RNAi-based approaches have been used as a therapeutic agent in the treatment of a variety of tumor types.37,38 In the present study, we injected MAP4-siRNA directly into the tumors formed by the MAP4-expressing ESCC cells in nude mice, and found that at day 10 after the first injection the mean tumor volume of the tested group was reduced by 86% as compared with the control. It has been reported that inhibition of VEGFA/VEGFR pathway is associated with reductions in tumor angiogenesis and with prolonged survival of patients.20 However, some cancer patients do not significantly prolong life after treatment by blocking VEGFA/VEGFR alone. In view that the cytoplasm of tumor cells in 50.7% (179/353) of ESCC tissues presented high expression of VEGFA or VEGFA/MAP4, we postulated whether VEGF in tumor cells targets to some intracellular signal molecule(s) besides the above VEGF/VEGFR-RAS/ERK signaling effect, When combined intratumoral injection of MAP4-siRNA with injection of Bevacizumab via tail vein, the inhibition to tumor growth was superior to that of Bevacizumab or MAP4-siRNA alone. It means when considering an administration of VEGF/VEGFR blocking, it should detect MAP4 expression to decide whether to add the inhibition of MAP4. In other words, for the patients with tumor highly coexpressing MAP4 and VEGF, a simultaneous blocking to both MAP4 and VEGF/VEGFR might be needed. In summary, our current work reveals a novel mechanism of tumor cell invasion and migration in ESCC, involving an activation of MAP4-ERK-VEGF-ERK pathway. We show that MAP4 is of prognostic and therapeutic relevance. Elevated expression of MAP4 protein is an independent indicator for shorter survival of the patients with ESCC. As an important regulator of ERK signaling, MAP4 may serve as a candidate molecular target for ESCC therapy. For ESCC patients with high expression of both MAP4 and VEGF, a combinational inhibition of MAP4 and VEGF probably improves the effectiveness of treatment compared with the blocking of VEGF alone. MATERIALS AND METHODS Cell culture and tissue specimens The human ESCC cell lines KYSE150 and KYSE180 were provided by Dr Shimada (Kyoto University) and Eca109 was obtained from the cell bank of type culture collection of Chinese academy of sciences (Shanghai Institute of Cell Biology, Chinese Academy of Sciences, Shanghai, China). The cell lines were cultured in RPMI 1640 medium with 10% fetal bovine serum (Invitrogen, San Diego, CA, USA), penicillin (100 U/ml) and streptomycin (100 mg/ml). Fresh ESCC tissues were procured from surgical resection specimens collected by the Department of Pathology at the Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College (CAMS & PUMC), Beijing, China. All patients received no treatment before surgery and signed informed consent forms of the Cancer Hospital, CAMS & PUMC for sample collection. This study was approved by the Ethics Committee/ Institutional Review Board of the Cancer Institute/Hospital, Peking Union Medical College and Chinese Academy of Medical Sciences (No. NCC2013RE-025).

SiRNA synthesis and plasmid construction The siRNA sequences against human MAP4, VEGFA, VEGFR1, ERK1/2 and nonsilencing were chemically synthesized by Genepharma (Shanghai, China; Supplementary Table S2). The small hairpin RNAs were synthesized by Invitrogen (Shanghai, China). The pLKO.1-MAP4-small hairpin RNA (sh-MAP4) and pLKO.1-scramble-small hairpin RNA (sh-scramble) were generated by inserting the corresponding double-stranded oligonucleotides into pLKO.1-puro lentiviral vector. The MAP4-expressing vector GV230-MAP4 was constructed by GeneChem (Shanghai, China). And the VEGFA-expressing © 2016 Macmillan Publishers Limited

plasmid pCS2vegf121 and pCS2vegf165 were acquired from Nathan Lawson (Addgene plasmid 22416 and 22417, Cambridge, MA, USA).

Gene transfection Cells were transfected with siRNA or plasmid vectors using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. Cells were harvested 48 h after transfection.

Conditioned media When the KYSE150 cells were grown to about 85% confluence, the medium was substituted for that without serum. After continued to incubate for 24 h, the cultures was collected and the supernatants were ultra-filtered with an Amicon Ultra-10 K device (Millipore, Temecula, CA, USA) at 5000 r.p.m./4 °C for 30 min. The concentration media was removed from Amicon into 1.5 ml sterile tubes and stored in − 20 °C.

Cell matrigel invasion and migration assays Cells were seeded on a fibronectin-coated polycarbonate membrane insert in a Transwell apparatus (Costar, Flintshire, UK). RPMI 1640 containing 20% fetal bovine serum (Invitrogen) was added to the lower chamber. After incubation for 40 (for Eca109) or 30 h (KYSE150) at 37 °C, the insert was washed with phosphate-buffered saline, and cells on the top surface of the insert were removed by wiping with a cotton swab. For the Matrigel chemoinvasion assay, the procedure was similar to the haptotactic cell migration assay, except that the Transwell membrane was coated with 300 ng/ml Matrigel (BD Biosciences, San Jose, CA, USA). The cells were stained with 0.4% crystal and counted in three random fields at × 200 magnification.

Wound-healing assay Cells were seeded into 60-mm dishes. When the cells were grown to confluence, three scrape wounds were made for each sample. The cells were photographed at 0, 10 and 30 h for Eca109, and 0, 10 and 24 h for KYSE150.

Co-immunoprecipitation The cells were washed with phosphate-buffered saline and lysed with lysis buffer (Roche Immunoprecipitation kit) for 1 h at 4 °C. 50 μl of the homogeneous protein G-agarose suspension was added to the sample and incubated for at least 3 h at 4 °C to reduce non-specific binding. After removing the beads, the supernatant was supplemented with 5 mg of the anti-MAP4 (Proteintech, Rosemont, IL, USA, 11229-1-AP) or anti-ERK1/2 (Cell Signaling, Danvers, MA, USA, #9102) antibody followed by incubation overnight at 4 °C. Protein G-agarose was then added to the immunoprecipitation mixture and the incubation was continued overnight at 4 °C. The immunoprecipitates were collected by centrifugation and washed with the wash buffer. Loading buffer was added, and the agarose was boiled and subjected to western blot analysis.

Luciferase assay The luciferase reporter assays were conducted according to the manufacturer's instructions (Promega, Madison, WI, USA). Each sample was carried out in duplicate, and the experiment was repeated at least three times. The transfection efficiency was measured by co-transfection with a Renilla luciferase expression plasmid pRL-SV40 (Promega). These data were presented as the ratio of firefly luciferase activity to Renilla luciferase activity. The results were presented as the mean ± s.e.m.

Chromatin immunoprecipitation A ChIP assay was performed with the ChIP kit (Active Motif). Chromatin samples were immunoprecipitated with an anti–phospho-c-Jun antibody (Cell Signaling, #3270). Anti-rabbit IgG (Santa Cruz, Dallas, TX, USA) was used as a negative control. Precipitated DNA was amplified by PCR using VEGFA-promoter region primers. Non-immunoprecipitated chromatin fragments were used as an input control.

Immunohistochemical analysis Immunohistochemical analysis was performed as described previously.39,40 The samples were incubated with anti-human MAP4 antibody and antihuman VEGFA (Proteintech, 19003-1-AP) overnight at 4 °C followed by incubation with biotinylated secondary antibody (Santa Cruz). The signals Oncogene (2016) 1 – 11

MAP4 is a potential therapeutic target for esophageal cancer Y-Y Jiang et al

10 were visualized by using PV-9000 and DAB kit (Zhong Shan, Beijing). The staining intensity of MAP4 and VEGFA were graded as the scales: 0 (negative), 1 (weak), 2 (moderate) as low expression and 3 (strong) as high expression.

Xenograft assays in nude mice Ten, 6–8 weeks, female BALB/c mice were s.c. injected (for tumor growth assay) and nine, 6–8 weeks, female NOD/SCID mice were tail vein injected (for tumor metastasis assay) with 1 × 106 cells transduced with sh-MAP4 or sh-scramble lentivirus per animal. The BALB/c mice were killed at day 18 and NOD/SCID mice at day 49 after injection. The animals were examined for s.c. tumor growth and metastases development. Immunohistochemistry was performed on 5-mm sections of paraffin-embedded s.c. tumors. The metastasis nodules in lung tissues were fixed in Bouin's solution, embedded in paraffin, cut into 5-mm sections and stained with hematoxylin and eosin. Before the animals were killed, mice blood was collected for measuring serum VEGFA by a commercial ELISA kit (Quantikine, Minneapolis, MN, USA, R&D systems). The absorption was read at 450 nm (reference) in a microplate reader.

Inhibition of tumor growth assay in vivo Invivofectamine-MAP4-siRNA complex and invivofectamine-non-silencingsiRNA complex were prepared according to manufacturer’s instructions (Life Technology, Carlsbad, CA, USA). siRNA (250 μl) stock solution (3 mg/ ml) was mixed with 250 μl of complex buffer. Invivofectamine reagent (500 μl) was added into the mixture and incubated for 30 min at 50 °C. The mixture was then added to the Float-A-Lyzer dialysis device (Thermo Scientific, Waltham, MA, USA) and incubated at room temperature for 2 h in 1 l of phosphate-buffered saline. The volume was adjusted to the desired concentration with phosphate-buffered saline. The mixture was injected via s.c. into the mice tumors that formed by the MAP4-expressing ESCC cells. The animal experiments of this study were approved by the Ethics Committee/Institutional Review Board (for experimental animals) of the Cancer Institute/Hospital, Peking Union Medical College and Chinese Academy of Medical Sciences (No. NCC2013A014).

Statistical analysis All statistical analyses were performed using SPSS 15.0 statistical program (SPSS Inc., Chicago, IL, USA). The correlation between MAP4 and VEGFA expression levels in primary ESCC tissues was analyzed by using the Pearson correlation test. Associations between protein expression and clinico-pathological parameters were assessed by the Χ2 test. For survival analyses, Kaplan–Meier survival curves were constructed, and differences were tested by the log-rank test. Multiple Cox proportional hazards regression was carried out to identify the independent factors with a significant impact on patient survival. All tests of significance were set at Po0.05.

CONFLICT OF INTEREST The authors declare no conflict of interest.

ACKNOWLEDGEMENTS This work was supported by National Natural Science Foundation of China (81330052 and 81321091) and National High-Tech R&D Program of China (2012AA02A503 and 2012AA020206).

REFERENCES 1 He YT, Hou J, Chen ZF, Qiao CY, Song GH, Meng FS et al. Decrease in the esophageal cancer incidence rate in mountainous but not level parts of Cixian County, China, over 29 years. Asian Pac J Cancer Prev 2005; 6: 510–514. 2 Luo ML, Shen XM, Zhang Y, Wei F, Xu X, Cai Y et al. Amplification and overexpression of CTTN (EMS1) contribute to the metastasis of esophageal squamous cell carcinoma by promoting cell migration and anoikis resistance. Cancer Res 2006; 66: 11690–11699. 3 Permana S, Hisanaga S, Nagatomo Y, Iida J, Hotani H, Itoh TJ. Truncation of the projection domain of MAP4 (microtubule-associated protein 4) leads to attenuation of microtubule dynamic instability. Cell Struct Funct 2005; 29: 147–157.

Oncogene (2016) 1 – 11

4 Kremer BE, Haystead T, Macara IG. Mammalian septins regulate microtubule stability through interaction with the microtubule-binding protein MAP4. Mol Biol Cell 2005; 16: 4648–4659. 5 Hu JY, Chu ZG, Han J, Dang YM, Yan H, Zhang Q et al. The p38/MAPK pathway regulates microtubule polymerization through phosphorylation of MAP4 and Op18 in hypoxic cells. Cell Mol Life Sci 2010; 67: 321–333. 6 Ookata K, Hisanaga S, Bulinski JC, Murofushi H, Aizawa H, Itoh TJ et al. Cyclin B interaction with microtubule-associated protein 4 (MAP4) targets p34cdc2 kinase to microtubules and is a potential regulator of M-phase microtubule dynamics. J Cell Biol 1995; 128: 849–862. 7 Chang W, Gruber D, Chari S, Kitazawa H, Hamazumi Y, Hisanaga S et al. Phosphorylation of MAP4 affects microtubule properties and cell cycle progression. J Cell Sci 2001; 114: 2879–2887. 8 Murphy M, Hinman A, Levine AJ. Wild-type p53 negatively regulates the expression of a microtubule-associated protein. Genes Dev 1996; 10: 2971–2980. 9 Ou Y, Zheng X, Gao Y, Shu M, Leng T, Li Y et al. Activation of cyclic AMP/PKA pathway inhibits bladder cancer cell invasion by targeting MAP4-dependent microtubule dynamics. Urol Oncol 2014; 32: 47.e21–47.e28. 10 Cantley LC. The phosphoinositide 3-kinase pathway. Science 2002; 296: 1655–1657. 11 Lee WJ, Chen WK, Wang CJ, Lin WL, Tseng TH. Apigenin inhibits HGF-promoted invasive growth and metastasis involving blocking PI3K/Akt pathway and beta 4 integrin function in MDA-MB-231 breast cancer cells. Toxicol Appl Pharmacol 2008; 226: 178–191. 12 Monami G, Gonzalez EM, Hellman M, Gomella LG, Baffa R, Iozzo RV et al. Proepithelin promotes migration and invasion of 5637 bladder cancer cells through the activation of ERK1/2 and the formation of a paxillin/FAK/ERK complex. Cancer Res 2006; 66: 7103–7110. 13 Honma N, Genda T, Matsuda Y, Yamagiwa S, Takamura M, Ichida T et al. MEK/ERK signaling is a critical mediator for integrin-induced cell scattering in highly metastatic hepatocellular carcinoma cells. Lab Invest 2006; 86: 687–696. 14 Galliher AJ, Schiemann WP. Src phosphorylates Tyr284 in TGF-beta type II receptor and regulates TGF-beta stimulation of p38 MAPK during breast cancer cell proliferation and invasion. Cancer Res 2007; 67: 3752–3758. 15 Zhu B, Shi S, Ma YG, Fan F, Yao ZZ. Lysophosphatidic acid enhances human hepatocellular carcinoma cell migration, invasion and adhesion through P38 MAPK pathway. Hepatogastroenterology 2012; 59: 785–789. 16 Patel V, Marsh CA, Dorsam RT, Mikelis CM, Masedunskas A, Amornphimoltham P et al. Decreased lymphangiogenesis and lymph node metastasis by mTOR inhibition in head and neck cancer. Cancer Res 2011; 71: 7103–7112. 17 Fruchon S, Kheirallah S, Al Saati T, Ysebaert L, Laurent C, Leseux L et al. Involvement of the Syk-mTOR pathway in follicular lymphoma cell invasion and angiogenesis. Leukemia 2012; 26: 795–805. 18 Gitay-Goren H, Soker S, Vlodavsky I, Neufeld G. The binding of vascular endothelial growth factor to its receptors is dependent on cell surface-associated heparin-like molecules. J Biol Chem 1992; 267: 6093–6098. 19 Park JE, Keller GA, Ferrara N. The vascular endothelial growth factor (VEGF) isoforms: differential deposition into the subepithelial extracellular matrix and bioactivity of extracellular matrix-bound VEGF. Mol Biol Cell 1993; 4: 1317–1326. 20 Huang S, Robinson JB, Deguzman A, Bucana CD, Fidler IJ. Blockade of nuclear factor-kappaB signaling inhibits angiogenesis and tumorigenicity of human ovarian cancer cells by suppressing expression of vascular endothelial growth factor and interleukin 8. Cancer Res 2000; 60: 5334–5339. 21 Chen HX, Gore-Langton RE, Cheson BD. Clinical trials referral resource: Current clinical trials of the anti-VEGF monoclonal antibody bevacizumab. Oncology (Williston Park) 2001; 15: 1017 1020, 1023-1016. 22 Enzinger PC, Mayer RJ. Esophageal cancer. N Engl J Med 2003; 349: 2241–2252. 23 Parysek LM, Asnes CF, Olmsted JB. MAP 4: occurrence in mouse tissues. J Cell Biol 1984; 99: 1309–1315. 24 Xu YB, Du QH, Zhang MY, Yun P, He CY. Propofol suppresses proliferation, invasion and angiogenesis by down-regulating ERK-VEGF/MMP-9 signaling in Eca-109 esophageal squamous cell carcinoma cells. Eur Rev Med Pharmacol Sci 2013; 17: 2486–2494. 25 Jiang G, Cao F, Ren G, Gao D, Bhakta V, Zhang Y et al. PRSS3 promotes tumour growth and metastasis of human pancreatic cancer. Gut 2010; 59: 1535–1544. 26 Deng Z, Sui G, Rosa PM, Zhao W. Radiation-induced c-Jun activation depends on MEK1-ERK1/2 signaling pathway in microglial cells. PLoS One 2012; 7: e36739. 27 Nagy JA, Vasile E, Feng D, Sundberg C, Brown LF, Detmar MJ et al. Vascular permeability factor/vascular endothelial growth factor induces lymphangiogenesis as well as angiogenesis. J Exp Med 2002; 196: 1497–1506. 28 Gerber HP, Dixit V, Ferrara N. Vascular endothelial growth factor induces expression of the antiapoptotic proteins Bcl-2 and A1 in vascular endothelial cells. J Biol Chem 1998; 273: 13313–13316.

© 2016 Macmillan Publishers Limited

MAP4 is a potential therapeutic target for esophageal cancer Y-Y Jiang et al

11 29 Gerber HP, McMurtrey A, Kowalski J, Yan M, Keyt BA, Dixit V et al. Vascular endothelial growth factor regulates endothelial cell survival through the phosphatidylinositol 3'-kinase/Akt signal transduction pathway. Requirement for Flk-1/ KDR activation. J Biol Chem 1998; 273: 30336–30343. 30 Neufeld G, Cohen T, Gengrinovitch S, Poltorak Z. Vascular endothelial growth factor (VEGF) and its receptors. FASEB J 1999; 13: 9–22. 31 Ferrara N, Gerber HP, LeCouter J. The biology of VEGF and its receptors. Nat Med 2003; 9: 669–676. 32 Shibuya M. Differential roles of vascular endothelial growth factor receptor-1 and receptor-2 in angiogenesis. J Biochem Mol Biol 2006; 39: 469–478. 33 Kappas NC, Zeng G, Chappell JC, Kearney JB, Hazarika S, Kallianos KG et al. The VEGF receptor Flt-1 spatially modulates Flk-1 signaling and blood vessel branching. J Cell Biol 2008; 181: 847–858. 34 Weigand M, Hantel P, Kreienberg R, Waltenberger J. Autocrine vascular endothelial growth factor signalling in breast cancer. Evidence from cell lines and primary breast cancer cultures in vitro. Angiogenesis 2005; 8: 197–204. 35 Lee TH, Seng S, Sekine M, Hinton C, Fu Y, Avraham HK et al. Vascular endothelial growth factor mediates intracrine survival in human breast carcinoma cells through internally expressed VEGFR1/FLT1. PLoS Med 2007; 4: e186.

36 Darrington E, Zhong M, Vo BH, Khan SA. Vascular endothelial growth factor A, secreted in response to transforming growth factor-beta1 under hypoxic conditions, induces autocrine effects on migration of prostate cancer cells. Asian J Androl 2012; 14: 745–751. 37 Brock A, Krause S, Li H, Kowalski M, Goldberg MS, Collins JJ et al. Silencing HoxA1 by intraductal injection of siRNA lipidoid nanoparticles prevents mammary tumor progression in mice. Sci Transl Med 2014; 6: 217ra212. 38 Tekedereli I, Alpay SN, Akar U, Yuca E, Ayugo-Rodriguez C, Han HD et al. Therapeutic silencing of Bcl-2 by systemically administered siRNA nanotherapeutics inhibits tumor growth by autophagy and apoptosis and enhances the efficacy of chemotherapy in orthotopic xenograft models of ER (-) and ER (+) breast cancer. Mol Ther Nucleic Acids 2013; 2: e121. 39 Feng YB, Lin DC, Shi ZZ, Wang XC, Shen XM, Zhang Y et al. Overexpression of PLK1 is associated with poor survival by inhibiting apoptosis via enhancement of survivin level in esophageal squamous cell carcinoma. Int J Cancer 2009; 124: 578–588. 40 Zhang Y, Feng YB, Shen XM, Chen BS, Du XL, Luo ML et al. Exogenous expression of Esophagin/SPRR3 attenuates the tumorigenicity of esophageal squamous cell carcinoma cells via promoting apoptosis. Int J Cancer 2008; 122: 260–266.

Supplementary Information accompanies this paper on the Oncogene website (http://www.nature.com/onc).

© 2016 Macmillan Publishers Limited

Oncogene (2016) 1 – 11

migration and a potential therapeutic target in esophageal squamous cell carcinoma.

Cell invasion and migration significantly contribute to tumor metastasis. Microtubule-associated protein 4 (MAP4) protein is one member of microtubule...
4MB Sizes 3 Downloads 7 Views