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Cofilin-phosphatase slingshot-1L (SSH1L) is over-expressed in pancreatic cancer (PC) and contributes to tumor cell migration
Cancer Letters 360 (2015) 171–176
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Cancer Letters j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / c a n l e t
Original Articles
Cofilin-phosphatase slingshot-1L (SSH1L) is over-expressed in pancreatic cancer (PC) and contributes to tumor cell migration Yufeng Wang a, Yasuhiro Kuramitsu a,*, Takao Kitagawa a, Byron Baron a, Shigefumi Yoshino b, Shin-Ichiro Maehara c, Yoshihiko Maehara c, Masaaki Oka b, Kazuyuki Nakamura a,d a Departments of Biochemistry and Functional Proteomics, Digestive Surgery of Applied Molecular Bioscience, Yamaguchi University Graduate School of Medicine, Ube, Japan b Digestive Surgery of Applied Molecular Bioscience, Yamaguchi University Graduate School of Medicine, Ube, Japan c Department of Surgery and Science, Graduate School of Medical Science, Kyusyu University, 3-1-1 Maidashi Higashiku, Fukuokashi, Fukuoka, Japan d Centre of Clinical Laboratories, Tokuyama Medical Association Hospital, Shunan, Japan
A R T I C L E
I N F O
Article history: Received 6 June 2014 Received in revised form 31 January 2015 Accepted 3 February 2015 Keywords: Pancreatic cancer SSH1L Cell migration
A B S T R A C T
Slingshot-1L (SSH1L), a cofilin-phosphatase, plays a role in actin dynamics and cell migration by reactivating cofilin-1. However, the expression of SSH1L in malignant diseases is poorly understood. The overexpression of SSH1L in cancerous tissue compared to the matched surrounding non-cancerous tissues from patients with late stages (III–IV) of PC was detected in 90% (9/10) of cases by western blotting. The expression of SSH1L was shown to be upregulated in tumor cells from 10.7% (11/102) of patients with pancreatic cancer (PC) by immunohistochemistry (IHC). The positive rate of SSH1L in patients with PC at stage VI (TNM) categorized as grade 3 was of 50% (2/4) and 15% (6/40), respectively. Moreover, SSH1L expression was shown to be up-regulated in the PC cell lines (KLM1, PANC-1 and MIAPaCa-2) with high metastatic potential. Loss of SSH1L expression was associated with an increase in the phosphorylation of cofilin-1 at serine-3 and further inhibited cell migration (but not proliferation) in KLM1, PANC-1 and MIAPaCa-2. Actin polymerization inhibitor cytochalasin-D was sufficient to abrogate cell migration of PC without changing SSH1L expression. These results reveal that SSH1L is upregulated in a subset of PCs and that the SSH1L/cofilin-1 signal pathway is associated positively in PC with cell migration. Our study may thus provide potential targets to prevent and/or treat PC invasion and metastasis in patients with SSH1L-positive PC. © 2015 Elsevier Ireland Ltd. All rights reserved.
Introduction Pancreatic cancer (PC) has an extremely poor prognosis and the 5 year survival rates are less than 5% [1]. Because of rapid aggressiveness, most patients are only diagnosed when they are already terminally ill. PCs can spread to the abdomen, liver, lung, bone and brain, and it would have spread too much to be removed by surgery or treated by radiation therapy alone. Gemcitabine has been the firstline treatment for patients with metastatic PC for several decades, but Gemcitabine resistance is becoming increasingly troublesome during therapy [2,3]. Therefore, inhibition or prevention of tumor metastasis can be an important therapeutic strategy for PC. The actin cytoskeleton plays a central role in cell migration such as cell-substrate adhesion, protrusion, phagocytosis and cytokinesis [4]. Cofilin and actin depolymerization factor (ADF) are essential
* Corresponding author. Tel: +81 836222213; fax: +81 836222212. E-mail address: [email protected] (Y. Kuramitsu). http://dx.doi.org/10.1016/j.canlet.2015.02.015 0304-3835/© 2015 Elsevier Ireland Ltd. All rights reserved.
regulators of actin dynamics [4–6]. The Rho family, small GTP binding protein (Rac1)/p21/Cdc42/Rac1-activated kinase 1 (Pak1)/LIM kinase (LIMK1) signaling pathway controls F-actin turnover and cell protrusion through regulation of cofilin activity [7]. The LIMK1/ cofilin pathway is often activated in cancers, and plays an important role in tumor invasion and metastasis [8–11]. Normally upregulated neu-associated kinase (HUNK), a metastasis suppressor, blocks actin polymerization and metastasis in breast cancer by sustaining the phosphorylation and inactivation of cofilin-1 [12]. Moreover, researchers have revealed that the phosphorylation and activity of cofilin have a direct association with invasion and metastasis of mammary tumors [13,14]. Our recent report has shown that cofilin1 (but not cofilin-2) is upregulated in the cancerous tissues compared to the surrounding non-cancerous tissues from patients with PC [15]. Taken together, these data suggest that cofilin-1 may be a candidate for clinical application as a therapeutic target in PC for inhibiting or preventing tumor cell invasion or metastasis. Slingshot-1L (SSH1L), a cofilin phosphatase, reactivates cofilin through dephosphorylation of cofilin at serine-3 and stimulates
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cofilin-mediated actin turnover [16]. Insulin-induced membrane protrusion as well as dephosphorylation and activation of cofilin are mediated by SSH1L activation, a downstream target of phosphoinositide 3-kinase [17]. However as a cofilin regulator, SSH1L has a much lower profile in malignant diseases than LIMK1. In our current study, we showed that SSH1L was elevated in cancerous tissues compared to non-cancerous tissues from patients with PC and in cancer cell lines presenting a high metastatic potential. SSH1L may potentially induce PC cell invasion and metastasis through dephosphorylation and activation of cofilin-1. Thus, this is a novel report of the role played by SSH1L in malignancy. Materials and methods PC tissues and cell culture Ten tissue samples were randomly selected from patients with pancreatic cancer, who had undergone surgical resection at the Department of Surgery II, Yamaguchi University Hospital (Table 1). Written informed consent was obtained from all patients before surgery. Tissues were obtained immediately after surgery and stored at −80 °C until use. The study protocol was approved by the Institutional Review Board for human study of Yamaguchi University School of Medicine. PC cell lines, KLM1, KLM1-R, AsPC-1, BxPC-3, PK45p and PK59, were cultured in Roswell Park Memorial Institute 1640 (RPMI 1640; GIBCO, 05918, Gaithersburg, MD) and PANC-1 and
Table 1 Clinicopathological parameters of patients with pancreatic cancer (for WB). No.
Age (year)
Sex
TNM stage
Tumor grade
1 2 3 4 5 6 7 8 9 10
48 73 54 57 74 72 53 69 67 60
Female Male Male Male Female Male Male Female Female Male
IVa IVa IVa III IVa IVa IVb III IVa IVb
Moderately differentiated Moderately differentiated Moderately differentiated Moderately differentiated Poorly differentiated Well differentiated Well differentiated Moderately differentiated Moderately differentiated Moderately differentiated
MIAPaCa-2 were cultured in Dulbecco’s Modified Eagle’s medium (DMEM; GIBCO, 12100-046) medium supplemented with 10% heat-inactivated fetal bovine serum (FBS; GIBCO, 26140-079) and 2 mM of L-glutamine, at 37 °C, in a humidified 5% CO2– 95% air mixture. Human pancreatic duct epithelial (HPDE) cells were cultured in keratinocyte serum-free (KSF) medium supplemented by epidermal growth factor and bovine pituitary extract (Life Technologies, Inc., Grand Island, NY) [18]. Materials Cytochalasin D (sc-201442) was purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). SSH1L siRNA (sc-96081) was purchased from Santa Cruz Biotechnology Inc. The antibodies specific for actin (sc-1616) and ERK (sc-94) were purchased from Santa Cruz Biotechnology Inc. The antibody specific for Cofilin-1 (WH0001072) was purchased from Sigma Aldrich Inc. (St. Louis, MO). The antibody specific for p-cofilin-1 at serine-3 (#3313) was purchased from Cell Signaling Technology Inc. (Boston, MA). The antibody specific for SSH1L (ab76943) was purchased from Abcam Biochemicals (Cambridge, UK). Knockdown assay Cells were cultured at 37 °C, in a humidified 5% CO2–95% air mixture until the cells were 70% confluent. The cells were transfected with validated human SSH1L siRNA or control siRNA (sc-37007, Santa Cruz Biotechnology) by following an siRNA Transfection Protocol (Santa Cruz Biotechnology). Western blot Tissues or cells were lysed in lysis buffer (1% NP-40, 1 mM sodium vanadate, 1 mM PMSF, 50 mM Tris, 10 mM NaF, 10 mM EDTA, 165 mM NaCl, 10 μg/mL leupeptin, and 10 μg/mL aprotinin) on ice for 1 h. Suspensions were centrifuged at 15,000 × g for 30 min at 4 °C. The supernatants were collected and resolved by SDS–polyacrylamide gel and then transferred onto PVDF membrane (Immobilon-P; Millipore, Bedford, MA). The membrane was blocked with 5% skimmed milk in TBS for 1 h and then incubated with the appropriate primary antibody at 4 °C, overnight. The membrane was washed and incubated with a horseradish peroxidase (HRP)-conjugated secondary antibody for 1 h at room temperature. Signal bands were visualized by using a chemiluminescence reagent (Immunostar; Wako, Osaka, Japan). Immunofluorescence and confocal microscopy Cells were cultured on coverslips and fixed by 3.7% paraformaldehyde in phosphate buffered saline (PBS) for 30 min. Cells were washed with PBS 3 times, followed by permeabilization with 0.1% Triton X-100 for 15 min. These were then washed with
Fig. 1. Changes in SSH1L protein levels in human pancreatic cancer. (A) The SSH1L expression in pancreatic cancers and the matched surrounding non-cancerous tissues of patients (n = 10) was detected by western bolt analysis. NT, non-cancerous tissue; CT, cancerous tissue (B) A few foci of weakly positive normal cells are visible, but various cancerous tissues are not immunoreactive. Strong immunoreactivity was shown in the poorly differentiated (grade 3) and stage IV ductal adenocarcinoma.
Y. Wang et al./Cancer Letters 360 (2015) 171–176
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PBS 3 times and incubated in blocking solution (1% goat serum or 1% donkey serum in PBS with 0.1% Tween 20) for 1 h at room temperature. Subsequently, the coverslips were treated with a primary antibody in blocking solution overnight at 4 °C. They were then rinsed with PBS with 0.1% Tween 20 (PBS-T) 5 times and incubated with a secondary antibody for 1 h at room temperature. They were washed once with PBS-T and stained with 1.43 μM DAPI (4,6′-diamidino-2-phenylindole) for 5 min. The coverslips were washed with PBS-T twice and mounted face-down onto microscope slides with Fluoromount (Diagnostic BioSystems, Pleasanton, CA). Cells were analyzed by using a Laser Scan Confocal Microscope (LSM 510 META; Carl Zeiss, Mobicity, Australia). All parameters were kept constant within each experiment.
(SP1711, ECM Biosciences LLC, Kentucky, USA) was added and the sections were incubated with diaminobenzidine substrate as chromogen.
Immunohistochemistry (IHC)
Wound healing assay
The expression of SSH1L was detected by using the pancreatic cancer tissue microarray (PA2081a) from US Biomax, Inc. The pathological information of patients was shown on the product website (http://www.biomax.us/tissue -arrays/Pancreas/PA2081a). The sections were cut from formalin-fixed and paraffinembedded tissue blocks. Primary rabbit polyclonal anti-SSH1L (1/100) antibody
The cells were cultured in 96-well flat-bottom plates until the cells were 70% confluent. After an appropriate treatment, one wound was scratched through the cells with a sterile 200 μL pipette tip. Cells were observed using a fluorescence microscope (KEYENCE BZ-9000; Itasca, IL) and quantification was performed by Image J software.
Proliferation assay Cells were cultured in 96 well cell culture plates until they were 70% confluent. After an appropriate treatment, 20 μL of the MTS dye (Promega, Madison, WI) was added to each well of the plate and incubated for a further 1.5 h. Optical density (OD) was used to assess the cell viabilities and was read directly at 492 nm by a Model 550 reader (BIO-RAD, Hercules, CA).
Results and discussion Table 2 Pathological parameters of patients with pancreatic cancer by IHC (SSH1L). Patients
SSH1L positive
Total
Ratio
Cases TNM stage I-II III IV Metastasis Tumor grade 1–2 3 Unknown
11
102
10.7%
6 1 2 2
74 12 4 12
8.1% 8.3% 50% 16.6%
2 6 3
47 40 15
4.2% 15% 20%
Overexpression of SSH1L in human PC Our previous proteomic study has shown that cofilin-1 is upregulated in cancerous tissues compared to non-cancerous tissues from patients with PC [15]. However, little was known about the expression of cofilin-phosphatase SSH1L in PC, or other malignant diseases. Investigating the protein expression of SSH1L in PC, western blot analysis showed that the overexpression rate of SSH1L in cancerous tissues from patients with PC at late stages (III–IV) is as high as 90% (9/10) compared to the matched non-cancerous tissues (Fig. 1A). Moreover, a tissue microarray containing 102 PC patient samples was used for IHC analysis (Fig. 1B). The expression of SSH1L
Fig. 2. SSH1L expression is associated with cancer cells having a high metastatic potential. (A) SSH1L expression and its colocalization with actin were detected by immunofluorescent analysis using confocal microscopy in various PC cell lines. SSH1L, green; actin, red; DAPI, blue. (B) SSH1L expression was detected in the PC cell lines and HPDE cells by western blotting. The expression of actin was used to normalize the loading volume. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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was shown to be upregulated in tumor cells from 10.7% (11/102) of patients with PC; the positive rate of SSH1L in patients with PC at Stages I–II, III and IV was 8.1%, 8.3% and 50%, respectively; the positive rate of SSH1L in patients with PC categorized as grades 1–2, and 3 was 4.2% and 15%, respectively (Table 2). Protein kinase D (PKD) regulates the cofilin signaling network via phosphorylation of SSH1L at serines 937 and 978 [19]. Cucurbitacin-B-induced cofilin–actin rod formation and actin aggregation in melanoma cells are known to depend on SSH1L-mediated cofilin hyperactivation [20]. Cofilin is known to play a critical role in tumor cell migration, invasion and metastasis [8–11]. Based on the above data, SSH1L is reported for the first time to be upregulated in malignant tumor tissues in this
study and it may play an important role in the tumor spreading through the regulation of the cofilin signaling network in PC. SSH1L is associated with cancer cells having high metastatic potential One of the characteristics of pancreatic cancer is that it spreads so rapidly to other parts of the body that surgery is not an option for patients. PC KLM1 cells have a higher ability to form liver metastasis (at the rate of 70%) compared to PK-1 and PK-9 cells (rarely observable) in nude mice [21]. One hundred PC MIAPaCa-2 or PANC-1 cells (but not Capan-1, Capan-2, BxPC-3 or PL45) were sufficient to
Fig. 3. Loss of SSH1L increases the phosphorylation of cofilin-1 at serine-3 in PC cells. (A) The expressions of SSH1L, p-cofilin-1 and cofilin-1 were detected in KLM1 and KLM1-R cells by western blot analysis. (B) and (C) The phosphorylation of cofilin-1 was detected in PANC-1 or MIA PaCa-2 cells by western blot analysis 48 h after knockdown of SSH1L. The expression of actin was used to normalize the loading volume.
A
B PANC-1 (24 h)
Cont. **
400
KLM1 KLM1-R
300 200 100 0
Cell proliferation
D
Cont.
siSSH1L
2 1.5 1 0.5 0
Cell migration
Cell migration
24 h
PANC-1
400
MIAPaCa-2 (24 h)
si-SSH1L Cont. siSSH1L
***
300 200 100 0
Cont.
Cell migration
KLM1-R
E
Cont. siSSH1L
150 100
Cont. 2.5 2 1.5 1 0.5 0
si-SSH1L **
200
PANC-1
Cell proliferation
KLM1
C
50 0
MIAPaCa-2
siSSH1L
MIA PaCa-2
Fig. 4. Loss of SSH1L inhibits the migration of PC cells. (A) Cell migration was detected by wound healing assay for 24 h, and quantification was performed by Student’s t-test. (B) and (C) Cell migration in PANC-1 or MIA PaCa-2 cells was investigated by wound healing assay for 24 h after SSH1L was knocked down for 48 h, and quantifications were performed by Student’s t-test. **, P < 0.01; ***, P < 0.001. (D) and (E) Cell proliferation was detected in PANC-1 or MIA PaCa-2 cells after SSH1L was knocked down for 72 h by MTS assay (Student’s t-test).
Y. Wang et al./Cancer Letters 360 (2015) 171–176
form liver metastasis in NOG mice, and the incidence was 71.4% (5/7) and 37.5% (3/8), respectively [22]. BxPC-3 metastasis was barely observed in NOD/SCID mice [22]. These data indicate KLM1, PANC-1 and MIAPaCa-2 cells present high metastatic potentials in the liver. The expression of SSH1L was examined by immunofluorescence and western blot in eight types of PC cell line (Fig. 2A and B), and the results show higher expression levels of SSH1L in KLM1, PANC-1 and MIAPaCa-2 compared to the other PC cell lines as well as HPDE cells. SSH1L binds to F-actin at Trp-458 (close to the C-terminus of the phosphatase domain), an LHKACE motif in the N-terminal region, and an LKRSHS motif in the C-terminal region, resulting in activation by F-actin [23]. The immunofluorescent analysis showed that SSH1L was perfectly co-localized with F-actin in cultured KLM1, PANC-1 and MIAPaCa-2 cells (Fig. 2A), indicating the active role of SSH1L in these cells. These results indicate that SSH1L is associated with tumor cells having a high metastatic potential and it may function as a metastatic regulator in PC. SSH1L regulates phosphorylation of cofilin-1 and cell migration in PC cells Cofilin is inactivated and reactivated via phosphorylation modulation at Serine-3 by LIM kinase and Slingshot respectively [17]. Platelet-derived growth factor (PDGF) induces migration of human aortic smooth muscle cells through activation of the SSH1L/cofilin signaling pathway [24,25]. Our previous study suggested that the upregulation of cofilin-1 may be involved in the invasion and
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metastasis of PC [15]. Thus, it was necessary to validate the effects of SSH1L on the cofilin-1 phosphorylation and cell migration in PC. Here, we found that the expression of SSH1L was dramatically downregulated and accompanied by increased phosphorylation of cofilin-1 at Serine-3 in gemcitabine resistant-KLM1-R compared to its related sensitive-KLM1 PC cell line (Fig. 3A). We also demonstrated that the phosphorylation of cofilin-1 at serine-3 was significantly increased by SSH1L knockdown in PANC-1 and MIAPaCa-2 cells (Fig. 3B and C). These results indicated that cofilin1 activity was tightly associated with the expression of SSH1L in various PC cell lines. Moreover, to assess the effects of SSH1L on the migration of PC, a wound healing assay was performed after silencing SSH1L expression. After 24 h healing, the number of migrated cells in KLM1-R was significantly lower than KLM1 (Fig. 4A). Treatment of SSH1L siRNA significantly reduced cell migration of PANC-1 and MIAPaCa-2 compared to the control siRNA (Fig. 4B and C). However, knockdown of SSH1L did not exhibit an effect on the proliferation in PANC-1 and MIAPaCa-2 cells (Fig. 4D and E). Taken together, SSH1L expression is related to cofilin-1 activity and furthermore affects the migration of PC cells. Thus it possibly and potentially contributes to the invasion and metastasis of PC. Actin cytoskeleton inhibitor inhibits cell migration in KLM1 Based on the function of the SSH1L/cofilin signaling pathway on actin polymerization and dynamics [17,25], we further investigated if blocking of the actin cytoskeleton can inhibit PC cell
Fig. 5. Cytochalasin D inhibits the cell migration of PC KLM1 cells. (A) The colocalization of SSH1L and actin cytoskeleton was observed after treatment with 5 μM of cytochalasin D for 5 h in KLM1 cells by immunofluorescent analysis and confocal microscopy. (B) The migration of KLM1 cells was investigated by wound healing assay, 24 h after treatment with cytochalasin D for 5 h. (C) The expressions of SSH1L, p-cofilin-1 and cofilin-1 were detected in KLM1 cells after treatment with cytochalasin D for 5 h by western blot analysis. The expression of actin was used to normalize the loading volume.
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migration. Cytochalasin D is an inhibitor of the actin cytoskeleton that binds to the F-actin polymer and prevents further addition of actin monomers [26,27]. Cytochalasin D has also been reported to inhibit the actin–cofilin interaction and reduce cofilin phosphorylation [27,28]. Here, we also showed that cytochalasin D had an effect on the phosphorylation of cofilin-1 (Fig. 5C). The actin cytoskeleton as well as cell migration were significantly blocked after KLM1 cells were exposed to the indicated concentrations of Cytochalasin D (Fig. 5A and B). The expression of SSH1L and its colocalization with actin were not affected by the treatment of cytochalasin D (Fig. 5A and C). These results provide further evidence that SSH1L modulation of PC cell migration may be through controlling cofilinmediated actin polymerization and dynamics. Moreover, we provide new insight into SSH1L localization, as a fraction of the population of this protein resided in the nuclei of KLM1 cells without the colocalization of actin, as determined by the use of confocal microscopy (Fig. 5A). These data suggest that SSH1L might play a role in transcription regulation signaling. In conclusion, SSH1L may play an important role in the tumor progression of PC. High expression level of SSH1L has frequently been observed in tumor cells from patients with PC at late stages, and it is associated with the cancer having a high metastatic potential in the current study. We also demonstrated that SSH1L is related to cofilin-1 activity and moreover affects the migration of PC cells. Therefore, our study suggests that SSH1L may be a potential target to prevent and/or treat PC invasion and metastasis. Acknowledgements We thank Dr. Junko Akada and Dr. Kazuhiro Tokuda for discussion regarding this paper. We thank Dr. Nobuaki Suzuki and Dr. Tomio Ueno for providing us the pancreatic cancer tissues. This work was supported in part by Grants-in-Aid from the Ministry of Education, Science, Sports, and Culture of Japan (no. 24501352 to Yasuhiro Kuramitsu). Immunoblot detection by LAS-1000 was done at the Gene Research Centre of Yamaguchi University.
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Cancer Letters j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / c a n l e t
Original Articles
Cofilin-phosphatase slingshot-1L (SSH1L) is over-expressed in pancreatic cancer (PC) and contributes to tumor cell migration Yufeng Wang a, Yasuhiro Kuramitsu a,*, Takao Kitagawa a, Byron Baron a, Shigefumi Yoshino b, Shin-Ichiro Maehara c, Yoshihiko Maehara c, Masaaki Oka b, Kazuyuki Nakamura a,d a Departments of Biochemistry and Functional Proteomics, Digestive Surgery of Applied Molecular Bioscience, Yamaguchi University Graduate School of Medicine, Ube, Japan b Digestive Surgery of Applied Molecular Bioscience, Yamaguchi University Graduate School of Medicine, Ube, Japan c Department of Surgery and Science, Graduate School of Medical Science, Kyusyu University, 3-1-1 Maidashi Higashiku, Fukuokashi, Fukuoka, Japan d Centre of Clinical Laboratories, Tokuyama Medical Association Hospital, Shunan, Japan
A R T I C L E
I N F O
Article history: Received 6 June 2014 Received in revised form 31 January 2015 Accepted 3 February 2015 Keywords: Pancreatic cancer SSH1L Cell migration
A B S T R A C T
Slingshot-1L (SSH1L), a cofilin-phosphatase, plays a role in actin dynamics and cell migration by reactivating cofilin-1. However, the expression of SSH1L in malignant diseases is poorly understood. The overexpression of SSH1L in cancerous tissue compared to the matched surrounding non-cancerous tissues from patients with late stages (III–IV) of PC was detected in 90% (9/10) of cases by western blotting. The expression of SSH1L was shown to be upregulated in tumor cells from 10.7% (11/102) of patients with pancreatic cancer (PC) by immunohistochemistry (IHC). The positive rate of SSH1L in patients with PC at stage VI (TNM) categorized as grade 3 was of 50% (2/4) and 15% (6/40), respectively. Moreover, SSH1L expression was shown to be up-regulated in the PC cell lines (KLM1, PANC-1 and MIAPaCa-2) with high metastatic potential. Loss of SSH1L expression was associated with an increase in the phosphorylation of cofilin-1 at serine-3 and further inhibited cell migration (but not proliferation) in KLM1, PANC-1 and MIAPaCa-2. Actin polymerization inhibitor cytochalasin-D was sufficient to abrogate cell migration of PC without changing SSH1L expression. These results reveal that SSH1L is upregulated in a subset of PCs and that the SSH1L/cofilin-1 signal pathway is associated positively in PC with cell migration. Our study may thus provide potential targets to prevent and/or treat PC invasion and metastasis in patients with SSH1L-positive PC. © 2015 Elsevier Ireland Ltd. All rights reserved.
Introduction Pancreatic cancer (PC) has an extremely poor prognosis and the 5 year survival rates are less than 5% [1]. Because of rapid aggressiveness, most patients are only diagnosed when they are already terminally ill. PCs can spread to the abdomen, liver, lung, bone and brain, and it would have spread too much to be removed by surgery or treated by radiation therapy alone. Gemcitabine has been the firstline treatment for patients with metastatic PC for several decades, but Gemcitabine resistance is becoming increasingly troublesome during therapy [2,3]. Therefore, inhibition or prevention of tumor metastasis can be an important therapeutic strategy for PC. The actin cytoskeleton plays a central role in cell migration such as cell-substrate adhesion, protrusion, phagocytosis and cytokinesis [4]. Cofilin and actin depolymerization factor (ADF) are essential
* Corresponding author. Tel: +81 836222213; fax: +81 836222212. E-mail address: [email protected] (Y. Kuramitsu). http://dx.doi.org/10.1016/j.canlet.2015.02.015 0304-3835/© 2015 Elsevier Ireland Ltd. All rights reserved.
regulators of actin dynamics [4–6]. The Rho family, small GTP binding protein (Rac1)/p21/Cdc42/Rac1-activated kinase 1 (Pak1)/LIM kinase (LIMK1) signaling pathway controls F-actin turnover and cell protrusion through regulation of cofilin activity [7]. The LIMK1/ cofilin pathway is often activated in cancers, and plays an important role in tumor invasion and metastasis [8–11]. Normally upregulated neu-associated kinase (HUNK), a metastasis suppressor, blocks actin polymerization and metastasis in breast cancer by sustaining the phosphorylation and inactivation of cofilin-1 [12]. Moreover, researchers have revealed that the phosphorylation and activity of cofilin have a direct association with invasion and metastasis of mammary tumors [13,14]. Our recent report has shown that cofilin1 (but not cofilin-2) is upregulated in the cancerous tissues compared to the surrounding non-cancerous tissues from patients with PC [15]. Taken together, these data suggest that cofilin-1 may be a candidate for clinical application as a therapeutic target in PC for inhibiting or preventing tumor cell invasion or metastasis. Slingshot-1L (SSH1L), a cofilin phosphatase, reactivates cofilin through dephosphorylation of cofilin at serine-3 and stimulates
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cofilin-mediated actin turnover [16]. Insulin-induced membrane protrusion as well as dephosphorylation and activation of cofilin are mediated by SSH1L activation, a downstream target of phosphoinositide 3-kinase [17]. However as a cofilin regulator, SSH1L has a much lower profile in malignant diseases than LIMK1. In our current study, we showed that SSH1L was elevated in cancerous tissues compared to non-cancerous tissues from patients with PC and in cancer cell lines presenting a high metastatic potential. SSH1L may potentially induce PC cell invasion and metastasis through dephosphorylation and activation of cofilin-1. Thus, this is a novel report of the role played by SSH1L in malignancy. Materials and methods PC tissues and cell culture Ten tissue samples were randomly selected from patients with pancreatic cancer, who had undergone surgical resection at the Department of Surgery II, Yamaguchi University Hospital (Table 1). Written informed consent was obtained from all patients before surgery. Tissues were obtained immediately after surgery and stored at −80 °C until use. The study protocol was approved by the Institutional Review Board for human study of Yamaguchi University School of Medicine. PC cell lines, KLM1, KLM1-R, AsPC-1, BxPC-3, PK45p and PK59, were cultured in Roswell Park Memorial Institute 1640 (RPMI 1640; GIBCO, 05918, Gaithersburg, MD) and PANC-1 and
Table 1 Clinicopathological parameters of patients with pancreatic cancer (for WB). No.
Age (year)
Sex
TNM stage
Tumor grade
1 2 3 4 5 6 7 8 9 10
48 73 54 57 74 72 53 69 67 60
Female Male Male Male Female Male Male Female Female Male
IVa IVa IVa III IVa IVa IVb III IVa IVb
Moderately differentiated Moderately differentiated Moderately differentiated Moderately differentiated Poorly differentiated Well differentiated Well differentiated Moderately differentiated Moderately differentiated Moderately differentiated
MIAPaCa-2 were cultured in Dulbecco’s Modified Eagle’s medium (DMEM; GIBCO, 12100-046) medium supplemented with 10% heat-inactivated fetal bovine serum (FBS; GIBCO, 26140-079) and 2 mM of L-glutamine, at 37 °C, in a humidified 5% CO2– 95% air mixture. Human pancreatic duct epithelial (HPDE) cells were cultured in keratinocyte serum-free (KSF) medium supplemented by epidermal growth factor and bovine pituitary extract (Life Technologies, Inc., Grand Island, NY) [18]. Materials Cytochalasin D (sc-201442) was purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). SSH1L siRNA (sc-96081) was purchased from Santa Cruz Biotechnology Inc. The antibodies specific for actin (sc-1616) and ERK (sc-94) were purchased from Santa Cruz Biotechnology Inc. The antibody specific for Cofilin-1 (WH0001072) was purchased from Sigma Aldrich Inc. (St. Louis, MO). The antibody specific for p-cofilin-1 at serine-3 (#3313) was purchased from Cell Signaling Technology Inc. (Boston, MA). The antibody specific for SSH1L (ab76943) was purchased from Abcam Biochemicals (Cambridge, UK). Knockdown assay Cells were cultured at 37 °C, in a humidified 5% CO2–95% air mixture until the cells were 70% confluent. The cells were transfected with validated human SSH1L siRNA or control siRNA (sc-37007, Santa Cruz Biotechnology) by following an siRNA Transfection Protocol (Santa Cruz Biotechnology). Western blot Tissues or cells were lysed in lysis buffer (1% NP-40, 1 mM sodium vanadate, 1 mM PMSF, 50 mM Tris, 10 mM NaF, 10 mM EDTA, 165 mM NaCl, 10 μg/mL leupeptin, and 10 μg/mL aprotinin) on ice for 1 h. Suspensions were centrifuged at 15,000 × g for 30 min at 4 °C. The supernatants were collected and resolved by SDS–polyacrylamide gel and then transferred onto PVDF membrane (Immobilon-P; Millipore, Bedford, MA). The membrane was blocked with 5% skimmed milk in TBS for 1 h and then incubated with the appropriate primary antibody at 4 °C, overnight. The membrane was washed and incubated with a horseradish peroxidase (HRP)-conjugated secondary antibody for 1 h at room temperature. Signal bands were visualized by using a chemiluminescence reagent (Immunostar; Wako, Osaka, Japan). Immunofluorescence and confocal microscopy Cells were cultured on coverslips and fixed by 3.7% paraformaldehyde in phosphate buffered saline (PBS) for 30 min. Cells were washed with PBS 3 times, followed by permeabilization with 0.1% Triton X-100 for 15 min. These were then washed with
Fig. 1. Changes in SSH1L protein levels in human pancreatic cancer. (A) The SSH1L expression in pancreatic cancers and the matched surrounding non-cancerous tissues of patients (n = 10) was detected by western bolt analysis. NT, non-cancerous tissue; CT, cancerous tissue (B) A few foci of weakly positive normal cells are visible, but various cancerous tissues are not immunoreactive. Strong immunoreactivity was shown in the poorly differentiated (grade 3) and stage IV ductal adenocarcinoma.
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PBS 3 times and incubated in blocking solution (1% goat serum or 1% donkey serum in PBS with 0.1% Tween 20) for 1 h at room temperature. Subsequently, the coverslips were treated with a primary antibody in blocking solution overnight at 4 °C. They were then rinsed with PBS with 0.1% Tween 20 (PBS-T) 5 times and incubated with a secondary antibody for 1 h at room temperature. They were washed once with PBS-T and stained with 1.43 μM DAPI (4,6′-diamidino-2-phenylindole) for 5 min. The coverslips were washed with PBS-T twice and mounted face-down onto microscope slides with Fluoromount (Diagnostic BioSystems, Pleasanton, CA). Cells were analyzed by using a Laser Scan Confocal Microscope (LSM 510 META; Carl Zeiss, Mobicity, Australia). All parameters were kept constant within each experiment.
(SP1711, ECM Biosciences LLC, Kentucky, USA) was added and the sections were incubated with diaminobenzidine substrate as chromogen.
Immunohistochemistry (IHC)
Wound healing assay
The expression of SSH1L was detected by using the pancreatic cancer tissue microarray (PA2081a) from US Biomax, Inc. The pathological information of patients was shown on the product website (http://www.biomax.us/tissue -arrays/Pancreas/PA2081a). The sections were cut from formalin-fixed and paraffinembedded tissue blocks. Primary rabbit polyclonal anti-SSH1L (1/100) antibody
The cells were cultured in 96-well flat-bottom plates until the cells were 70% confluent. After an appropriate treatment, one wound was scratched through the cells with a sterile 200 μL pipette tip. Cells were observed using a fluorescence microscope (KEYENCE BZ-9000; Itasca, IL) and quantification was performed by Image J software.
Proliferation assay Cells were cultured in 96 well cell culture plates until they were 70% confluent. After an appropriate treatment, 20 μL of the MTS dye (Promega, Madison, WI) was added to each well of the plate and incubated for a further 1.5 h. Optical density (OD) was used to assess the cell viabilities and was read directly at 492 nm by a Model 550 reader (BIO-RAD, Hercules, CA).
Results and discussion Table 2 Pathological parameters of patients with pancreatic cancer by IHC (SSH1L). Patients
SSH1L positive
Total
Ratio
Cases TNM stage I-II III IV Metastasis Tumor grade 1–2 3 Unknown
11
102
10.7%
6 1 2 2
74 12 4 12
8.1% 8.3% 50% 16.6%
2 6 3
47 40 15
4.2% 15% 20%
Overexpression of SSH1L in human PC Our previous proteomic study has shown that cofilin-1 is upregulated in cancerous tissues compared to non-cancerous tissues from patients with PC [15]. However, little was known about the expression of cofilin-phosphatase SSH1L in PC, or other malignant diseases. Investigating the protein expression of SSH1L in PC, western blot analysis showed that the overexpression rate of SSH1L in cancerous tissues from patients with PC at late stages (III–IV) is as high as 90% (9/10) compared to the matched non-cancerous tissues (Fig. 1A). Moreover, a tissue microarray containing 102 PC patient samples was used for IHC analysis (Fig. 1B). The expression of SSH1L
Fig. 2. SSH1L expression is associated with cancer cells having a high metastatic potential. (A) SSH1L expression and its colocalization with actin were detected by immunofluorescent analysis using confocal microscopy in various PC cell lines. SSH1L, green; actin, red; DAPI, blue. (B) SSH1L expression was detected in the PC cell lines and HPDE cells by western blotting. The expression of actin was used to normalize the loading volume. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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was shown to be upregulated in tumor cells from 10.7% (11/102) of patients with PC; the positive rate of SSH1L in patients with PC at Stages I–II, III and IV was 8.1%, 8.3% and 50%, respectively; the positive rate of SSH1L in patients with PC categorized as grades 1–2, and 3 was 4.2% and 15%, respectively (Table 2). Protein kinase D (PKD) regulates the cofilin signaling network via phosphorylation of SSH1L at serines 937 and 978 [19]. Cucurbitacin-B-induced cofilin–actin rod formation and actin aggregation in melanoma cells are known to depend on SSH1L-mediated cofilin hyperactivation [20]. Cofilin is known to play a critical role in tumor cell migration, invasion and metastasis [8–11]. Based on the above data, SSH1L is reported for the first time to be upregulated in malignant tumor tissues in this
study and it may play an important role in the tumor spreading through the regulation of the cofilin signaling network in PC. SSH1L is associated with cancer cells having high metastatic potential One of the characteristics of pancreatic cancer is that it spreads so rapidly to other parts of the body that surgery is not an option for patients. PC KLM1 cells have a higher ability to form liver metastasis (at the rate of 70%) compared to PK-1 and PK-9 cells (rarely observable) in nude mice [21]. One hundred PC MIAPaCa-2 or PANC-1 cells (but not Capan-1, Capan-2, BxPC-3 or PL45) were sufficient to
Fig. 3. Loss of SSH1L increases the phosphorylation of cofilin-1 at serine-3 in PC cells. (A) The expressions of SSH1L, p-cofilin-1 and cofilin-1 were detected in KLM1 and KLM1-R cells by western blot analysis. (B) and (C) The phosphorylation of cofilin-1 was detected in PANC-1 or MIA PaCa-2 cells by western blot analysis 48 h after knockdown of SSH1L. The expression of actin was used to normalize the loading volume.
A
B PANC-1 (24 h)
Cont. **
400
KLM1 KLM1-R
300 200 100 0
Cell proliferation
D
Cont.
siSSH1L
2 1.5 1 0.5 0
Cell migration
Cell migration
24 h
PANC-1
400
MIAPaCa-2 (24 h)
si-SSH1L Cont. siSSH1L
***
300 200 100 0
Cont.
Cell migration
KLM1-R
E
Cont. siSSH1L
150 100
Cont. 2.5 2 1.5 1 0.5 0
si-SSH1L **
200
PANC-1
Cell proliferation
KLM1
C
50 0
MIAPaCa-2
siSSH1L
MIA PaCa-2
Fig. 4. Loss of SSH1L inhibits the migration of PC cells. (A) Cell migration was detected by wound healing assay for 24 h, and quantification was performed by Student’s t-test. (B) and (C) Cell migration in PANC-1 or MIA PaCa-2 cells was investigated by wound healing assay for 24 h after SSH1L was knocked down for 48 h, and quantifications were performed by Student’s t-test. **, P < 0.01; ***, P < 0.001. (D) and (E) Cell proliferation was detected in PANC-1 or MIA PaCa-2 cells after SSH1L was knocked down for 72 h by MTS assay (Student’s t-test).
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form liver metastasis in NOG mice, and the incidence was 71.4% (5/7) and 37.5% (3/8), respectively [22]. BxPC-3 metastasis was barely observed in NOD/SCID mice [22]. These data indicate KLM1, PANC-1 and MIAPaCa-2 cells present high metastatic potentials in the liver. The expression of SSH1L was examined by immunofluorescence and western blot in eight types of PC cell line (Fig. 2A and B), and the results show higher expression levels of SSH1L in KLM1, PANC-1 and MIAPaCa-2 compared to the other PC cell lines as well as HPDE cells. SSH1L binds to F-actin at Trp-458 (close to the C-terminus of the phosphatase domain), an LHKACE motif in the N-terminal region, and an LKRSHS motif in the C-terminal region, resulting in activation by F-actin [23]. The immunofluorescent analysis showed that SSH1L was perfectly co-localized with F-actin in cultured KLM1, PANC-1 and MIAPaCa-2 cells (Fig. 2A), indicating the active role of SSH1L in these cells. These results indicate that SSH1L is associated with tumor cells having a high metastatic potential and it may function as a metastatic regulator in PC. SSH1L regulates phosphorylation of cofilin-1 and cell migration in PC cells Cofilin is inactivated and reactivated via phosphorylation modulation at Serine-3 by LIM kinase and Slingshot respectively [17]. Platelet-derived growth factor (PDGF) induces migration of human aortic smooth muscle cells through activation of the SSH1L/cofilin signaling pathway [24,25]. Our previous study suggested that the upregulation of cofilin-1 may be involved in the invasion and
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metastasis of PC [15]. Thus, it was necessary to validate the effects of SSH1L on the cofilin-1 phosphorylation and cell migration in PC. Here, we found that the expression of SSH1L was dramatically downregulated and accompanied by increased phosphorylation of cofilin-1 at Serine-3 in gemcitabine resistant-KLM1-R compared to its related sensitive-KLM1 PC cell line (Fig. 3A). We also demonstrated that the phosphorylation of cofilin-1 at serine-3 was significantly increased by SSH1L knockdown in PANC-1 and MIAPaCa-2 cells (Fig. 3B and C). These results indicated that cofilin1 activity was tightly associated with the expression of SSH1L in various PC cell lines. Moreover, to assess the effects of SSH1L on the migration of PC, a wound healing assay was performed after silencing SSH1L expression. After 24 h healing, the number of migrated cells in KLM1-R was significantly lower than KLM1 (Fig. 4A). Treatment of SSH1L siRNA significantly reduced cell migration of PANC-1 and MIAPaCa-2 compared to the control siRNA (Fig. 4B and C). However, knockdown of SSH1L did not exhibit an effect on the proliferation in PANC-1 and MIAPaCa-2 cells (Fig. 4D and E). Taken together, SSH1L expression is related to cofilin-1 activity and furthermore affects the migration of PC cells. Thus it possibly and potentially contributes to the invasion and metastasis of PC. Actin cytoskeleton inhibitor inhibits cell migration in KLM1 Based on the function of the SSH1L/cofilin signaling pathway on actin polymerization and dynamics [17,25], we further investigated if blocking of the actin cytoskeleton can inhibit PC cell
Fig. 5. Cytochalasin D inhibits the cell migration of PC KLM1 cells. (A) The colocalization of SSH1L and actin cytoskeleton was observed after treatment with 5 μM of cytochalasin D for 5 h in KLM1 cells by immunofluorescent analysis and confocal microscopy. (B) The migration of KLM1 cells was investigated by wound healing assay, 24 h after treatment with cytochalasin D for 5 h. (C) The expressions of SSH1L, p-cofilin-1 and cofilin-1 were detected in KLM1 cells after treatment with cytochalasin D for 5 h by western blot analysis. The expression of actin was used to normalize the loading volume.
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migration. Cytochalasin D is an inhibitor of the actin cytoskeleton that binds to the F-actin polymer and prevents further addition of actin monomers [26,27]. Cytochalasin D has also been reported to inhibit the actin–cofilin interaction and reduce cofilin phosphorylation [27,28]. Here, we also showed that cytochalasin D had an effect on the phosphorylation of cofilin-1 (Fig. 5C). The actin cytoskeleton as well as cell migration were significantly blocked after KLM1 cells were exposed to the indicated concentrations of Cytochalasin D (Fig. 5A and B). The expression of SSH1L and its colocalization with actin were not affected by the treatment of cytochalasin D (Fig. 5A and C). These results provide further evidence that SSH1L modulation of PC cell migration may be through controlling cofilinmediated actin polymerization and dynamics. Moreover, we provide new insight into SSH1L localization, as a fraction of the population of this protein resided in the nuclei of KLM1 cells without the colocalization of actin, as determined by the use of confocal microscopy (Fig. 5A). These data suggest that SSH1L might play a role in transcription regulation signaling. In conclusion, SSH1L may play an important role in the tumor progression of PC. High expression level of SSH1L has frequently been observed in tumor cells from patients with PC at late stages, and it is associated with the cancer having a high metastatic potential in the current study. We also demonstrated that SSH1L is related to cofilin-1 activity and moreover affects the migration of PC cells. Therefore, our study suggests that SSH1L may be a potential target to prevent and/or treat PC invasion and metastasis. Acknowledgements We thank Dr. Junko Akada and Dr. Kazuhiro Tokuda for discussion regarding this paper. We thank Dr. Nobuaki Suzuki and Dr. Tomio Ueno for providing us the pancreatic cancer tissues. This work was supported in part by Grants-in-Aid from the Ministry of Education, Science, Sports, and Culture of Japan (no. 24501352 to Yasuhiro Kuramitsu). Immunoblot detection by LAS-1000 was done at the Gene Research Centre of Yamaguchi University.
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