Myosin regulatory light chain phosphorylation is associated with leiomyosarcoma development

Biomedicine & Pharmacotherapy 92 (2017) 810–818

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Original article

Myosin regulatory light chain phosphorylation is associated with leiomyosarcoma development Hua-Shan Lia,1, Qian Lina,1, Jia Wua,1, Zhi-Hui Jianga , Jia-Bi Zhaob , Jian Panc , Wei-Qi Hea,* , Juan-Min Zhaa,* a Cambridge-Suda (CAM-SU) Genome Resource Center, Soochow University, and Department of Oncology, The First Affiliated Hospital of Soochow University, Suzhou 215123, China b Department of Pathology, The Third Affiliated Hospital of Soochow University, Changzhou 213004, China c Institute of Pediatrics, Children's Hospital of Soochow University, Suzhou 215003, China

A R T I C L E I N F O

Article history: Received 24 February 2017 Received in revised form 16 May 2017 Accepted 29 May 2017 Keywords: Myosin light chain Myosin light chain kinase Expression Leiomyosarcoma Proliferation

A B S T R A C T

Leiomyosarcoma is a rare malignant smooth muscle tumor which can be very unpredictable. Myosin II is involved in many functions, including cell contraction, migration, and adhesion. The phosphorylation of myosin regulatory light chain (MLC) by myosin light chain kinase (MLCK) determines the activity of Myosin II. However, it is still unclear whether MLC phosphorylation is involved in cell proliferation in leiomyosarcoma. In this study, we aimed to explore the role of MLCK-dependent MLC phosphorylation in leiomyosarcoma development. We found that the expression of MLCK, phosphorylated MLC, and Ki67 in leiomyosarcoma was significantly higher than in leiomyoma and adjacent normal smooth muscle cells. MLCK expression was significantly correlated with phosphorylated MLC level. Kaplan-Meier survival analysis revealed that patients with high expression of MLCK or phosphorylated MLC had shorter overall survival times compared with the patients with low expression of MLCK or phosphorylated MLC. In vitro studies revealed a causative link between MLC phosphorylation and cellular proliferation as expression of phosphomimetic MLC (T19D, S20D) increased cellular proliferation as assessed by Ki67 staining. In contrast, MLCK specific inhibitor reduced cellular proliferation. We concluded that MLCK, phosphorylated MLC and Ki67 were overexpressed in leiomyosarcoma. MLCK dependent MLC phosphorylation might be responsible for the high proliferative state in leiomyosarcoma. MLCK and phosphorylated MLC are potential prognostic indicators of leiomyosarcoma. © 2017 Elsevier Masson SAS. All rights reserved.

1. Introduction Soft tissue sarcomas are malignant tumors that develop in connective tissues. They account for
1% of all malignancies [1]. Leiomyosarcoma is an aggressive soft tissue sarcoma arises from smooth muscle cells, including uterine, gastrointestinal or other soft tissue origin. Leiomyosarcoma is the most common soft tissue sarcoma subtype, which accounts for
20% of soft tissue sarcomas

Abbreviations: ACTG2, smooth muscle gamma actin; CASQ2, calsequestrin 2; CFL2, human muscle cofilin 2; DAB, diaminobenzidine tetrahydrochloride; FBS, fetal bovine serum; MLC, myosin regulatory light chain; MLCK, myosin light chain kinase; PBS, phosphate buffered saline; SLMAP, sarcolemmal membrane associated protein. * Corresponding authors. E-mail addresses: [email protected] (W.-Q. He), [email protected] (J.-M. Zha). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.biopha.2017.05.139 0753-3322/© 2017 Elsevier Masson SAS. All rights reserved.

[2]. Currently, morphologic and immunohistochemical tools (e.g. actin and desmin immunostaining) are used for diagnosis of leiomyosarcoma [3]. However, the molecular pathogenesis of leiomyosarcoma is poorly understood. Effective molecular biomarkers are not currently available for the diagnosis, prognosis, or determination of treatments in leiomyosarcoma. Cellular proliferation is one fundamental characteristic of malignant tumor and leiomyosarcomas exhibit high proliferative activity [3] There are a few published studies regarding leiomyosarcoma and Ki67). As an index of cell proliferation, Ki67 is used as a predictive and prognostic marker of breast cancer [4,5], prostate cancer [6–8], and adrenalcortical carcinoma [9–12]. There are a few studies suggest that Ki67 is a prognostic factor for leiomyosarcoma [13,14]. Muscle contraction and actin cytoskeleton proteins, including smooth muscle gamma actin (ACTG2), calsequestrin 2 (CASQ2), human muscle cofilin 2 (CFL2), myosin light chain kinase (MLCK), and sarcolemmal membrane associated protein (SLMAP), are significantly enriched in leiomyosarcoma

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[1,15,16]. The contractile activity of myosin II is regulated by the phosphorylation of the myosin regulatory light chain (MLC) [17,18]. The amount and activity of myosin light chain kinase (MLCK) determine the phosphorylation level of MLC [19–21]. Targeted deletion of MLCK in mice demonstrates its central role in regulation of MLC phosphorylation and smooth muscle contraction [17,18]. In addition, MLCK-dependent MLC phosphorylation is a key regulator of many other cell functions, including cell migration, adhesion, and epithelial barrier regulation [22–26]. Interestingly, MLCK-dependent MLC phosphorylation is also necessary for tumor cell proliferation and division, and inhibition of MLCK by pharmacologic inhibitor blocks breast cancer cell growth [27–31]. In this study, we aimed to explore potential diagnostic and/or pronostic indicators of leiomyosarcoma. We performed immunohistochemistry on tissues from leiomyosarcoma samples to evaluate the expression of MLCK, phosphorylation MLC, and Ki67. We also tested cell proliferation with the expression of phosphomimetic MLC (T19D, S20D) in vitro, to determine whether phosphorylation of MLC was responsible for tumor cell proliferation. We identified increases in MLCK expression, MLC phosphorylation, and Ki67-positivity in leiomyosarcoma. Expression of phosphomimetic MLC increased cell proliferation. We suggested that expression of MLCK and phosphorylated MLC are potential prognostic indicators of leiomyosarcoma.

incubated in the retrieval buffer in a steamer for 20 mins for antigenic retrieval. Each slide was treated with 3% hydrogen peroxide for 5 min to eliminate endogenous peroxide. After washing with phosphate-buffered saline (PBS), each slide was incubated with MLCK (1:1000, Sigma, USA), phosphorylated MLC (1:100, Cell Signaling, USA), or Ki67 (1:200, Ruiying Biological, China) overnight at 4  C. Afterwards, the slides were washed with PBS and incubated with biotinylated second antibody for one hour at room temperature. The slides were finally developed with diaminobenzidine tetrahydrochloride (DAB) and stained with hematoxylin. All slides were assessed by two independent pathologists in a blinded manner. The expression of proteins was evaluated semiquantitatively based on staining intensity and percentages of positive stained cells. The intensity score was defined as 0, negtive; 1, weak; 2, moderate; and 3, strong; and the proportion score was defined as 0, 0–10%; 1, 11–25%; 2, 26–50%; 3, 51–75%; and 4, 76–100% positive cells. The final scores of protein expression were evaluated by the combination of staining intensity and distribution. For MLCK expression, scores of 8–12 were considered as high expression, while scores lower than 8 were considered as low expression. For phosphorylated MLC expression, 4–8 scores were considered as high expression, while scores lower than 4 were considered as low expression. Images were collected using an Olympus IX-73 microscope (Olympus, Japan).

2. Materials and methods

2.4. Cell culture and stable cell lines establishment

2.1. Patients and samples

SK-LMS-1 (ATCC) and HeLa cells were maintained in Modified Eagle’s Medium (DMEM) (Invitrogen, USA) containing 10% fetal bovine serum (FBS) (Gibco, USA). The medium was replaced every 2–3 days. The cDNA of human MLC and MLC (T19D, S20D) were cloned into a doxycycline inducible PiggyBac expression vector (pBH-EF1a) and verified by sequencing. The primers used for cloning are: MLC forward, 50 -GCC ACC ATG TCC AGC AAG CGG GCC AAA-30 ; MLC (T19D, S20D) forward, 50 - GCC ACC ATG TCC AGC AAG CGG GCC AAA GCC AAG ACC ACC AAG AAG CGG CCA CAG CGG GCC GAC GAC 30 ; Mutations were indicated with underlines. MLC and MLC (T19D, S20D) reverse, 50 - GTC GTC TTT ATC CTT GGC GC 30 . Expression plasmids were transfected into HeLa cells and stable expressing cells were selected by addition of 200 mg/ml of Hygromycin B into the medium.

Paraffin-embedded tissue blocks of 16 leiomyosarcoma, 16 leiomyoma, and 16 adjacent normal smooth muscle tissues were retrieved from the First Affiliated Hospital of Soochow University and the Third Affiliated Hospital of Soochow University between 2010 and 2016. For 16 leiomyosarcoma patients, there were 5 males and 11 females with a mean age of 56.4 years. 12 patients were classified as high/middle differentiated and 4 patients as low differentiated leiomyosarcoma according to American Joint Committee on Cancer (AJCC) Staging. Leiomyoma and normal smooth muscle tissues were from 16 female uterine myoma patients with a mean age of 48.7 years (Table 1). 2.2. Ethics statement

2.5. Cell proliferation assay This study was approved by the medical ethics committee of Soochow University. Written informed consent was obtained from the study participants. All methods were performed in accordance with the approved guidelines and regulations. 2.3. Immunohistochemistry (IHC) staining and microscopy Paraffin-embedded slides were deparaffinized in xylene and rehydrated with a graded ethanol series. The slides were then

SK-LMS-1 and HeLa cells were seeded with a density of 5  104 cells/well in 24-well plate. Cell growth was monitored by IncuCyte Live Cell Analysis System (Essen Bioscience, USA). Cells were cultured with medium containing ROCK inhibitor (Selleck,), Y27632 (Selleck), PKC inhibitor (Selleck), MLCK inhibitor ML-7 (Selleck), PIK (PIK, a membrane-permeant inhibitor of MLC kinase, sequence, RKKYKYRRK, synthesized by GL Biochem, Shanghai).

Table 1 Clinical features of patients with leimyosarcomas, leimyoma and normal smooth muscle tissues. Characteristics

Control No. (%)

Leimyoma No. (%)

Leiomyosarcoma No. (%)

Patient No. Gender Male Female Age (years) Differentiation Grade Low High/middle

16

16

16

0 16 48.7  12.9

0 16 48.7  12.9

5 11 56.4  17.0 4 12

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Cell proliferation was monitored by analyzing the occupied area (% confluence) of cell Images 60 or 72 h after seeding.

Fig. 1C–D), compared with normal or leiomyoma tissues. Interestingly, MLCK likely showed membrane localization in leiomyosarcoma patients.

2.6. Immunofluorescence staining and microscopy HeLa cells were plated on chamber slides (Lab-Tek, USA) and grown to
90% of confluency. Cells were fixed with 1% paraformaldehyde. Cells were washed with PBS and permeabilized with 0.1% Trion X-100 in PBS. After another around of PBS washes, cells were stained with anti-Ki67 antibody (Ruiying Biological, China), followed by species-specific secondary antisera conjugated to Alexa Fluor 594 (Invitrogen, USA), and Hoechst 33342 (Life Technologies, USA). Images were collected using an Olympus IX-73 microscope (Olympus, Japan). 2.7. Statistical analysis Student’s t-test was used to compare protein expression between different groups. Linear relationships were assessed by least squares regression analysis. Kaplan-Meier survival analysis (log-rand test) was used to evaluate the relationship between protein expression and the survival.

3.2. Increased expression of phosphorylated MLC in leiomyosarcoma patients Phosphorylated MLC was majorly found in cytoplasm of muscle fibers in uterine myoma and adjacent normal smooth muscle, and in leiomyosarcoma tissues (Fig. 2A–C). Phosphorylated MLC was intensely expressed in vascular smooth muscle cells in normal uterine tissues (Fig. 2A). The expression of phosphorylated MLC was significantly higher in patients with leiomyosarcoma compared with normal or leiomyoma patients (expression scores, normal, 1.3  0.5; leiomyoma, 1.2  0.5; leiomyosarcoma, 3.0  0.6; P–< 0.05 Fig. 2D). Similar to MLCK, phosphorylated MLC also showed membrane localization in leiomyosarcoma patients. The relationship between MLCK and phosphorylated MLC expression was examined. There was a positive correlation between MLCK and phosphorylated MLC level in leiomyosarcoma patients (R2 = 0.6199, P = 0.003; Fig. 3). 3.3. Increased expression of Ki67 in leiomyosarcoma patients

3. Results 3.1. Increased expression of MLCK in leiomyosarcoma patients Cytoplasmic distribution of MLCK was found in muscle fibers of uterine myoma and adjacent normal smooth muscle (expression scores, normal, 3.2  0.4; leiomyoma, 2.9  0.5, Fig. 1A–B). MLCK was intensely expressed in vascular smooth muscle cells in uterine myoma and adjacent normal uterine tissues (Fig. 1A-B). MLCK was widely expressed in cytoplasm of cells in leiomyosarcoma. The expression of MLCK is significantly higher in tissues from leiomyosarcoma patients (expression scores, 6.1 1.2, P < 0.05,

The expression of Ki67 was significantly higher in patients with leiomyosarcoma or leiomyoma patients compared with normal smooth muscle tissues (expression scores, normal, 1.8  0.3; leiomyoma, 2.9  0.4; leiomyosarcoma, 5.9  0.3; P < 0.01 Fig. 4). 3.4. Corrections of MLCK and phosphorylated MLC with the prognosis of leiomyosarcoma patients Kaplan-Meier survival analysis indicated that leiomyosarcoma patients with strong MLCK expression had significantly worse survival probability compared with those with weak MLCK

Fig. 1. Increased expression of MLCK in leiomyosarcoma. Representative immunohistochemistry staining of MLCK in normal control smooth muscle (A), leimyoma (B) and leiomyosarcoma (C). Scale bar, 200 mm. (D) Quantitative analysis of MLCK expression. Values are means  SD. Significant differences are indicated by: * P < 0.05 by two-tailed t-test.

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Fig. 2. Increased expression of phosphorylated MLC in leiomyosarcoma. Representative immunohistochemistry staining of MLCK in normal control smooth muscle (A), leimyoma (B) and leiomyosarcoma (C). Scale bar, 200 mm. (D) Quantitative analysis of phosphorylated MLC (pMLC) expression. Values are means  SD. Significant differences are indicated by: *P < 0.05 by two-tailed t-test.

established cervical cancer cell line [30]. HeLa cells were stably transfected with EGFP (MOCK), MLC-EGFP and MLC (T19D, S20D)EGFP expression constructs, and monitored by IncuCyte Live Cell Analysis System (Fig. 6A-C) 60 h after seeding. The confluence of cells expressing MLC (T19D, S20D)-EGFP was significantly higher than MOCK transfected or MLC-EGFP expressing cells (MOCK, 75.2  3.9%; MLC, 72.4  4.4%; phosphomimetic MLC, 90.0  5.2%, P < 0.05, Fig. 6D). Increased Ki67 expression was detected in MLC (T19D, S20D)EGFP expressing cells (MOCK, 2.4  0.2%; MLC, 2.8  0.3%; phosphomimetic MLC, 4.9  0.6%, P < 0.01, Fig. 7). 3.6. Inhibition of MLCK reduced proliferation of human SK-LMS-1 cells

Fig. 3. Expression of phosphorylated MLC is positively corrected with expression of MLCK in leiomyosarcoma. Correction analysis between the expression of phosphorylated MLC (pMLC) and MLCK in leimyosarcoma tissues. Scattergrams show the protein expression scores.

expression (Log-rank test: P < 0.05, Fig. 5A). Similarly, strong expression of phosphorylated MLC also conferred a poor prognosis, as leiomyosarcoma patients with elevated pMLC had significantly lower survival probability compared with those with weak phosphorylated MLC expression (Log-rank test: P < 0.05, Fig. 5B). 3.5. Expression of phosphomimetic MLC increased proliferation of human HeLa cells To determine whether the correlation between MLCK and pMLC expression had a causative relationship, we used a well-

MLC is majorly phosphorylated by MLCK. However, MLC phosphorylation can also be regulated by Protein Kinase C (PKC), Rho-associated kinase (ROCK) [19,32,33]. We then used pharmacologic inhibitors to study whether other kinases rather than MLCK were involved in cell proliferation. HeLa cells are originated from epithelial cell and may be different from leiomyosarcoma cells. Thus we also used a leiomyosarcoma cell line SK-LMS-1 in this experiment. We treated SK-LMS-1 cells with medium containing a ROCK specific inhibitor Y27632, a PKC specific inhibitor GF109203X, a MLCK specific inhibitor ML-7, and a peptide, membrane-permeant inhibitor of MLCK (PIK). The level of phosphorylated MLC was determined by immunostaining with anti-phosphorylated MLC antibody. ML-7 and PIK treatment significantly decreased the phosphorylated MLC expression in SK-LMS-1 cells (Fig. 8A–B). As shown in Fig. 8C, the confluence of SK-LMS-1 cells treated with ML-7 (10 mM) was significantly lower than untreated ones (76.1  4.2% vs. 92.7  1.4%, P < 0.01). PIK treatment also inhibited cell growth at the concentration of 100 mM (72.3  6.0%, P < 0.01). In contrast, Y27632 (10 mM, 90.2  2.0%) or GF109203X (1 mM, 92.1  3.0%) had no significant effect on cell growth. Cell confluence was monitored by IncuCyte Live Cell Analysis System

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Fig. 4. Increased expression of Ki67 in leimyoma and leiomyosarcoma. (B) Representative immunohistochemistry staining of Ki67 in normal control smooth muscle (A), leimyoma (B) and leiomyosarcoma (C). Scale bar, 200 mm. (D) Quantitative analysis of Ki67 expression. Values are means  SD. Significant differences are indicated by: *P < 0.01 by two-tailed t-test.

Fig. 5. Kaplan-Meier survival curves for leimyosarcoma patients with different expression of MLCK and phosphorylated MLC. (A) The overall survival of leimyosarcoma patients with high/low MLCK expression. Log-rank test: *P < 0.05. (B) The overall survival of leimyosarcoma patients with high/low phosphorylated MLC (pMLC) expression. Log-rank test: *P < 0.05.

72 h after seeding. To further prove the phosphorylation of MLC can enhance cell proliferation, we treated HeLa cells stably transfected with MLC-EGFP and phosphomimetic MLC with MLCK inhibitors. Both ML-7 and PIK decreased the growth of MOCK and MLC-EGFP expressed cells. However, these inhibitors did not inhibit the growth of phosphomimetic MLC expressed cells (Fig. 8D). Cell confluence was monitored 60 h after seeding. 4. Discussion The molecular pathogenesis of leiomyosarcoma is largely unknown. It is a major interest in identifying prognostic indicators of leiomyosarcoma to facilitate the development of targeted therapies. Cell proliferation is the basic component of malignant disease and a large tumor could arise by excess proliferation and resistance to cell death [3]. For example, cell proliferation maker, Ki67, is used as a prognostic marker of breast cancer [4,5], prostate

cancer [6–8], and adrenalcortical carcinoma [9–12]. Recently, it was reported that some muscle and cytoskeleton related proteins were highly enriched in leiomyosarcoma [15,16]. In our study, we aimed to explore potential muscle and cytoskeleton related prognostic indicators of leiomyosarcoma. We confirmed high proliferative state of leiomyosarcoma indicated by Ki67 staining of proliferative cells. Interestingly, MLCK and phosphorylated MLC, which are central to smooth muscle contraction, were overexpressed in leiomyosarcoma. Besides the key role in regulation of smooth muscle contraction, MLCK dependent MLC phosphorylation is also necessary for many other cell functions, including tumor cell proliferation and division [28,29]. Overexpression of MLCK expression increases cell proliferation and related to the development of liver tumorgenesis [29], while MLCK inhibition causes breast cancer cell growth inhibition [30]. MLCK regulates S phase entry in cell cycle progression in hepatocytes [27]. It is suggested that MLC

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Fig. 6. Expression of phosphomimetic MLC increased cell growth of human HeLa cells. Representative images of MOCK transfected cells (A) or cells stably transfected with MLC-EGFP (B) and MLC (T19D, S20D)-EGFP (C). Scale bar, 200 mm. (D) Quantitative analysis of cell confluences of MOCK, MLC-EGFP, and MLC (T19D, S20D) transfected cells. Cell confluences were recorded 72 h after seeding. Values are means  SD. Significant differences are indicated by: *P < 0.05 by two-tailed t-test.

Fig. 7. Expression of phosphomimetic MLC increased Ki67 positive cells. Representative Hoechst 33342 (blue) and Ki67 (red) stained images of MOCK transfected cells (A) or cells stably transfected with MLC-EGFP (B) and MLC (T19D, S20D)-EGFP (C). Scale bar, 200 mm. (D) Quantitative analysis of Ki67 expressing cells of MOCK, MLC-EGFP, and MLC (T19D, S20D) transfected HeLa cells. Values are means  SD. Significant differences are indicated by: **P < 0.01 by two-tailed t-test. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

phosphorylation causes interaction between myosin and actin, thus controlling cytoskeleton activity and regulates cell division and growth [31]. Moreover, a recent study showed that decreased expression of MLCK in smooth muscle tissues (MLCK intronic CArG knock-out mice) was associated with decreased myosin light chain

phosphorylation and impaired small intestine smooth muscle cell proliferation [34]. The knock-out mice had a shorter length of small intestine and fewer positively stained smooth muscle cells in small intestine, demonstrating the MLCK and MLC phosphorylation are

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Fig. 8. Inhibition of MLCK kinase activity reduced cell growth of human SK-LMS-1 cells. (A) Representative Hoechst 33342 (blue) and phosphorylated MLC (red) stained images of SK-LMS-1 cells. Scale bar, 200 mm. (B) Quantitative analysis of phosphorylated MLC level of SK-LMS-1 cells untreated, or treated with ROCK inhibitor Y27632 (10 mM), PKC inhibitor GF109203X (1 mM), MLCK inhibitor ML-7 (10 mM) and PIK (100 mM). (C) Quantitative analysis of cell confluences of SK-LMS-1 cells untreated, or treated with these inhibitors. (D) Quantitative analysis of cell confluences of MOCK, MLC-EGFP, and MLC (T19D, S20D) transfected HeLa cells treated with these inhibitors. Values are means  SD. Significant differences are indicated by: *P < 0.05, **P < 0.01 by two-tailed t-test. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

required for the regulation of smooth muscle cell proliferation [34]. To further determine whether MLCK dependent MLC phosphorylation is important for cell proliferation, we expressed the phosphomimetic MLC in HeLa cells. We showed that expression of

phosphomimetic MLC increased cell growth rate and Ki67 positive cells. MLCK is responsible for the phosphosrylation of MLC. However, MLC phosphorylation can also be regulated by several Ca2+-independent kinases including Protein Kinase C (PKC) and Rho-associated kinase (ROCK) [19,32,33]. Our study by using of

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pharmacologic inhibitors demonstrated that inhibition of MLCK, rather than PKC or ROCK, significantly block the tumor cell growth. Our results are supported by several recent findings. Davidson et al. identified genes participating in pathways related to the actin cytoskeleton and RhoA signaling in leiomyosarcoma. Interestingly, MLCK is overexpressed in leiomyosarcoma [16]. Beck et al. performed gene expression patterns on 271 cases of leiomyosarcoma, and identified five muscle contraction and actin cytoskeleton proteins (ACTG2, CASQ2, CFL2, MLCK, and SLMAP), are highly expressed in leiomyosarcoma [1,15]. Francis et al. performed gene expression profiling on 40 leiomyosarcoma samples. They also reported high expression levels of a group of muscle related genes, including MLCK, in leiomyosarcoma [35]. We analyzed MLCK expression in the microarray data published [36,37], and identified a 9.2-fold increase of MLCK mRNA expression in leiomyosarcoma tissue specimens compared with normal tissues. In conclusion, we observed that MLCK dependent phosphorylation of MLC is significantly higher in leiomyosarcoma than in leiomyoma and adjacent normal smooth muscle cells. The phosphorylation of MLC may be responsible for the high proliferative state in leiomyosarcoma. Patients with high expression of MLCK and phosphorylation level of MLC had shorter overall survival times compared with these with low expression of MLCK and phosphorylation level of MLC. Thus MLCK and phosphorylated MLC could be potential prognostic indicators of leiomyosarcoma.

[6]

[7]

[8]

[9]

[10]

[11] [12]

[13]

[14]

Competing interests The authors declare no competing financial interests.

[15]

Author contributions Juan-Min Zha, Wei-Qi He, and Jian Pan designed and performed the experiments, and wrote the manuscript; Hua-Shan Li, Qian-Lin, Jia Wu, Zhi-Hui Jiang, and Jia-Bi Zhao conducted the main experiments. All authors read and approved the manuscript. Acknowledgments We thank Dr. Matthew A. Odenwald at The University of Chicago for his discussion and suggestion in the preparation of the manuscript. This work was supported by National Natural Science Foundation of China (grant number 81200620, 81470804, and 31401229, 81570125), the Natural Science Foundation of Jiangsu Province (grant number BK20140319), The Research Innovation Program for College Graduates of Jiangsu Province (grant number KYLX16-0116), Advanced Research Projects of Soochow University (grant number SDY2015B06), and Crohn’s & Colitis Foundation of America (CCFA) Research Fellowship Award (grant number 310801).

[16]

[17]

[18]

[19] [20] [21]

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