Kindlin-2 in pancreatic stellate cells promotes the progression of pancreatic cancer

Cancer Letters 390 (2017) 103e114

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Original Article

Kindlin-2 in pancreatic stellate cells promotes the progression of pancreatic cancer Naoki Yoshida a, Atsushi Masamune a, *, Shin Hamada a, Kazuhiro Kikuta a, Tetsuya Takikawa a, Fuyuhiko Motoi b, Michiaki Unno b, Tooru Shimosegawa a a b

Division of Gastroenterology, Tohoku University Graduate School of Medicine, Sendai, Japan Division of Hepato-Biliary-Pancreatic Surgery, Tohoku University Graduate School of Medicine, Sendai, Japan

a r t i c l e i n f o

a b s t r a c t

Article history: Received 18 September 2016 Received in revised form 2 December 2016 Accepted 10 January 2017

Pancreatic stellate cells (PSCs) play a pivotal role in pancreatic fibrosis associated with pancreatic ductal adenocarcinoma (PDAC). Kindlin-2 is a focal adhesion protein that regulates the activation of integrins. This study aimed to clarify the role of kindlin-2 in PSCs in pancreatic cancer. Kindlin-2 expression in 79 resected pancreatic cancer tissues was examined by immunohistochemical staining. Kindlin-2knockdown immortalized human PSCs were established using small interfering RNA. Pancreatic cancer cells were treated with conditioned media of PSCs, and the cell proliferation and migration were examined. SUIT-2 pancreatic cancer cells were subcutaneously injected into nude mice alone or with PSCs and the size of the tumors was monitored. Kindlin-2 expression was observed in PDAC and the peritumoral stroma. Stromal kindlin-2 expression was associated with shorter recurrence-free survival time after R0 resection. Knockdown of kindlin-2 resulted in decreased proliferation, migration, and cytokine expression in PSCs. The PSC-induced proliferation and migration of pancreatic cancer cells were suppressed by kindlin-2 knockdown in PSCs. In vivo, co-injection of PSCs increased the size of the tumors, but this effect was abolished by kindlin-2 knockdown in PSCs. In conclusion, kindlin-2 in PSCs promoted the progression of pancreatic cancer. © 2017 Elsevier B.V. All rights reserved.

Keywords: Desmoplasia FERMT2 Integrin Pancreatic fibrosis Stroma

Introduction Pancreatic ductal adenocarcinoma (PDAC) is characterized by a highly malignant phenotype including rapid progression, early metastasis, and a limited response to chemotherapy and radiotherapy [1,2]. Abundant stroma is a characteristic feature of pancreatic cancer, and the stroma accounts for up to 90% of the total tumor volume [3]. It has been established that activated pancreatic stellate cells (PSCs) play a pivotal role in the development of pancreatic fibrosis in pancreatic cancer [4e10]. They not only produce extracellular matrix components, but also dynamically

Abbreviations: BrdU, 5-bromo-20 -deoxyuridine; CM, conditioned media; CP, chronic pancreatitis; EMT, epithelialemesenchymal transition; HPF, high power field; IPA, Ingenuity Pathways Analysis; OD, optical density; PDAC, pancreatic ductal adenocarcinoma; PSCs, pancreatic stellate cells; siRNA, small interfering RNA; SMA, smooth muscle actin; SE, standard error. * Corresponding author. Division of Gastroenterology, Tohoku University Graduate School of Medicine, 1-1 Seiryo-machi, Aoba-ku, Sendai 980-8574 Japan. Fax: þ81 22 717 7177. E-mail address: [email protected] (A. Masamune). http://dx.doi.org/10.1016/j.canlet.2017.01.008 0304-3835/© 2017 Elsevier B.V. All rights reserved.

interact with other cell types to constitute the cancer-conditioned microenvironment. Although it is still controversial, previous in vitro and in vivo studies have suggested that PSCs might promote the progression of pancreatic cancer through bi-directional interactions with pancreatic cancer cells [4e10]. The interactions between pancreatic cancer cells and PSCs are in part regulated by cytokines and growth factors such as fibroblast growth factor-2, TGF-b1, and IL-6 [4,8e10]. Kindlins are evolutionarily conserved focal adhesion proteins that interact with integrins and regulate the activation of integrins through binding to the integrin b cytoplasmic tails [11e14]. There are three types of kindlins: kindlin-1 (also known as FERMT1), kindlin-2 (also known as FERMT2 or MIG2), and kindlin-3 (also known as FERMT3). Kindlin-1 is mainly expressed in epithelial cells, kindlin-2 is expressed in wide variety of cell types but is absent in blood cells, and kindlin-3 is expressed in hematopoietic cells [14]. Kindlins are essential regulators of integrin signaling and integrin-mediated cell adhesion to the extracellular matrix [12,15]. Aberrant expression of kindlins has been reported in several types of cancer (reviewed in Refs. [14,16]). There have been a few studies

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reporting a role of kindlins in pancreatic cancer. Mahawithitwong et al. [17] reported that kindlin-1 was heterogeneously expressed in PDAC cells, while normal ductal epithelial cells and stromal cells showed no expression. Two previous studies have reported kindlin2 expression in pancreatic cancer, but with conflicting results [18,19]. Mahawithitwong et al. [18] reported that kindlin-2 was highly expressed in the stroma of PDAC but not in PDAC cells or normal duct epithelial cells. Zhan et al. [19] reported high expression of kindlin-2 in PDAC cells. They also reported that TGF-binduced kindlin-2 promoted the growth, migration and invasion of PDAC cells. This study aimed to clarify the role of kindlin-2 in pancreatic cancer, focusing on PSCs. Materials and methods Ethics This study was approved by the Ethics Committee of Tohoku University Graduate School of Medicine (article #: 2015-1-612 and 2016-1-92). Written informed consent was obtained from all patients. All animal procedures were performed in accordance with the National Institutes of Health guidelines for the care and use of laboratory animals, and the regulations for animal experiments and related activities at Tohoku University (article#: 2014-199 and 2014-200). Materials Mouse anti-kindlin-2 antibody was purchased from Merck Millipore (Billerica, MA). Rabbit anti-a-smooth muscle actin (a-SMA) antibody was purchased from Abcam (Cambridge, MA). Cell culture plates and dishes were purchased from Thermo Fisher Scientific (Waltham, MA). Other reagents were purchased from SigmaeAldrich (St. Louis, MO) unless specifically described. Immunohistochemical staining and evaluation The pancreas tissues were removed from 79 patients undergoing operation for pancreatic cancer at Tohoku University Hospital between January 2011 and December 2013. The clinicopathological characteristics of the 79 patients are shown in Table 1. Normal pancreatic tissues were taken from areas of peripheral tissue away from the tumor. In addition, the pancreas tissues were obtained from patients undergoing operation for chronic pancreatitis (CP). Tissue samples collected at the time of surgery were fixed in 10% paraformaldehyde overnight, embedded in paraffin wax, and cut into 4-mm sections. Immunohistochemical staining for kindlin-2 was

performed as previously described [20] using mouse anti-kindlin-2 antibody and streptavidinebiotineperoxidase complex detection kit (Histofine Kit; Nichirei, Tokyo, Japan). Immunostained sections were viewed to evaluate the percentage of kindlin-2-positive cells in PDAC or stromal cells as previously described [21]. All sections were viewed independently by two researchers to evaluate the intensity of staining. A consensus justification based on discussion was recorded. The degree of staining was defined as follows: low, <50% positive cells were found; high, 50% positive cells were found. Overall and recurrence-free survival time from the date of surgery was analyzed on December 31st, 2015. Double immunofluorescent staining Double immunofluorescent staining for kindlin-2 and a-SMA was performed as previously described [20]. Briefly, tissue sections were deparaffinized and rehydrated in PBS. Following antigen retrieval with the target retrieval solution (Agilent Technologies, Santa, Clara, CA), the slides were blocked with 3% BSA and incubated with mouse anti-kindlin-2 antibody and rabbit anti-a-SMA antibody overnight at 4  C. After washing, the slides were incubated for 1 h with Alexa Fluor546-labeled goat anti-rabbit IgG antibody (Thermo Fisher Scientific) and Alexa Fluor488-labeled donkey anti-mouse IgG antibody (Thermo Fisher Scientific). After washing, the cells were analyzed for fluorescence using an all-in-one type fluorescent microscope (BioZero BZ-9000; Keyence, Osaka, Japan). Nuclear counterstaining was performed using 40 , 6-diamidino-2-phenylindole. Cell culture Human pancreatic cancer cell lines except for SUIT-2 were obtained from American Type Culture Collection (Manassas, VA). SUIT-2 cells were obtained from Japanese Collection of Research Bioresources (Ibaraki, Osaka, Japan). Cells were maintained in RPMI 1640 medium supplemented with 10% FBS, penicillin sodium, and streptomycin sulfate. Primary human PSCs were isolated from the resected pancreas tissues of patients undergoing surgery for pancreatic cancer (hPSC1, hPSC2, and hPSC3) or CP ((hPSC4 and hPSC5) as previously described [22]. Primary human PSCs were maintained in Ham's F-12/DMEM (1:1) supplemented with 10% heatinactivated FBS, penicillin sodium, and streptomycin sulfate. The immortalized human PSC line hPSC21-S/T was established by retrovirus-mediated gene transfer of simian virus 40 T antigen and human telomerase reverse transcriptase into human primary PSCs [23]. The hPSC21-S/T cells expressed typical stellate cell markers including a-SMA, vimentin, type I collagen, and glial fibrillary acidic protein [10]. hPSC21-S/T cells were maintained in DMEM supplemented with 10% FBS, 100 U/mL penicillin and 100 mg/mL streptomycin. The conditioned media (CM) of hPSC21-S/T cells was harvested following 72-h incubation, centrifuged at 3000 revolution/min for 10 min, filtered through 0.22mm filters and stored at 80  C until use. Establishment of kindlin-2-knockdown immortalized human PSCs

Table 1 Clinicopathological characteristics of patients with PDAC (n ¼ 79). Characteristics

Values

Age (range), yrs Sex (Male/Female) Histologic grade 1 2 3 T-UICC category T1 T2 T3 T4 pN category pN0 pN1 Lymphatic invasion ly1þ/2þ ly3þ Vascular invasion v1þ/2þ v3þ Perineural invasion ne1þ/2þ ne3þ Adjuvant chemotherapy No Yes Radiation therapy No Yes

65.0 ± 9.1 (41e85) 51 (64.6%)/28 (35.4%) 9 (11.4%) 63 (79.7%) 7 (8.9%) 2 (2.5%) 7 (8.9%) 68 (86.1%) 2 (2.5%) 19 (24.1%) 60 (75.9%) 51 (64.6%) 28 (35.4%) 40 (50.6%) 39 (49.3%)

The small interfering RNA (siRNA)-expressing vectors were established by cloning the synthesized and annealed oligonucleotides into the pBAsi-hU6-Pur vector (Takara Bio, Otsu, Japan). The siRNA-expressing vector that targets 50 GAATCAATCAGCTTTACGA-30 corresponding to the nucleotide sequence of human kindlin-2 (NCBI accession number NM_006832.2) was transfected to hPSC21-S/T cells using the 4D-Nucleofector (Lonza, Basel, Switzerland). The control hPSC21-S/T cells were established by the introduction of a siRNA-expressing vector that targets 50 -TCTTAATCGCGTATAAGGC-30 , which does not correspond to any known human mRNA. The next day, the medium was replaced with fresh medium containing 1 mg/ ml puromycin. The puromycin-containing medium was replaced every 3 days and, after 14 days, puromycin-resistant colonies had developed. The kindlin-2knockdown hPSC21-S/T cell lines (designated as H-1 cells and H-2 cells) and the control cell line (designated as H-C cells) were cloned by limiting dilution in 96-well culture plates. Transient knockdown of kindlin-2 in primary human PSCs Primary human PSCs were transfected with the siRNA against kindlin-2 (#4392420; Ambion, Thermo Fisher Scientific) or the Negative Control No. 1 siRNA (#4390843; Ambion, Thermo Fisher Scientific) using the 4D-Nucleofector according to the manufacturer's instruction. The next day, the medium was replaced with fresh normal growth medium, and CM was prepared after additional 72-h incubation as described above. RNA isolation and quantitative real-time PCR

32 (40.5%) 47 (59.5%) 9 (11.4%) 70 (88.6%) 68 (86.1%) 11 (13.9%)

Quantitative real-time PCR was performed using Taqman® Universal PCR Master Mix II and detected using the StepOnePlus Real-Time PCR System (Thermo Fisher Scientific) [24]. Primers and probes were predesigned by the manufacturer (Thermo Fisher Scientific). The assay ID numbers were as follows: Hs00235033_m1 for kindlin-2 (FERMT2), Hs00605382_ml for chemokine (C-X-C motif) ligand 1 (CXCL1), Hs00601975_ml for CXCL2, Hs00234140_m1 for C-C motif chemokine ligand 2 (CCL2), Hs00174097_ml for IL-1b, Hs00985639_m1 for IL-6, Hs00174103_ml for IL-8, Hs00195591_ml for snail, Hs00185584_m1 for vimentin, and Hs03929097_g1 for

N. Yoshida et al. / Cancer Letters 390 (2017) 103e114 GAPDH. The expression of each mRNA was calculated from a sample standard curve. The values of target mRNAs in each sample were normalized by the respective GAPDH expression level. mRNA microarray and data analysis Total RNAs including microRNAs were extracted using the microRNA RNeasy preparation kit (Qiagen, Valencia, CA) according to the manufacturer's instructions. The mRNA microarray (SurePrint G3 Human Gene Expression v3 8  60K Microarray Kit; Agilent Technologies) was used to identify differentially expression between the kindlin-2-knockdown hPSC21-S/T (H-1) cells and the control (H-C) cells. Data analysis was performed using the GeneSpring GX software version 13.0 (Agilent Technologies). Data analysis and filtering were performed as described previously [10]. For the identification of up- or down-regulated genes, we calculated the Z-scores and ratios (non-log scaled fold-change) from the normalized signal intensities of each probe. We used the following criteria for up-regulated genes: Z-score 2.0 and ratio 1.5fold; and down-regulated genes: Z-score 2.0 and ratio 0.66. The heatmap was generated using the R software with the gplots and gtools packages (http://www.rproject.org/). We used a hierarchical clustering method to sort the genes. We used Ingenuity Pathways Analysis (IPA) (Ingenuity Systems, Redwood City, CA) to map the molecular pathways and networks populated by the predicted miRNA targets. This analysis lists genes in the context of known biological responses and regulatory networks as well as other higher-order response pathways. The IPA Database is a resource of published literature on gene functions and interactions. The version 27216297 (Release Date: 16 March, 2016) was used.

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membrane was incubated with mouse anti-kindlin-2 antibody overnight at 4  C. After incubation with peroxidase-conjugated anti-mouse IgG antibody (Cell Signaling Technologies, Beverly, MA), proteins were visualized by use of an ECL kit (GE Healthcare, Chalfont St. Giles, UK). The levels of GAPDH and a-tubulin were determined in a similar manner. Densitometric analysis was performed using the Image J software (http://rsweb.nih.gov/ij/ondex.html). Cell proliferation assay Cell proliferation was assessed using a commercial kit (Roche Applied Science) based on the measurement of 5-bromo-20 -deoxyuridine (BrdU) incorporation during DNA synthesis. Cells were labeled with BrdU for 3 h at 37  C. Cells were fixed and incubated with peroxidase-conjugated anti-BrdU antibody. Then the peroxidase substrate 3,30 ,5,50 -tetramethylbenzidine was added, and BrdU incorporation was quantified by optical density (OD)370-OD492. Migration assay Cell migration was assessed by scratch assay and a modified two-chamber migration assay. For a scratch assay, cells were grown to confluence in a 12-well culture plate. A monolayer of the cells was scratched with sterile 200-ml pipette tips. Cellular migration toward the scratched area was viewed. For a modified two-chamber assay, 1  104 cells were seeded into the culture inserts with 8-mm pores placed in a 24-well companion plate and the migration during 18 h toward the lower chamber was evaluated. Cells in the upper chamber were carefully removed using a cotton plug, and cells at the bottom of the membrane were fixed, stained with crystal violet and counted in 5 randomly chosen high power fields (HPFs) (100 magnification).

Western blotting Cells were lysed in sodium dodecyl sulfate buffer, and total cellular proteins (~100 mg) were fractionated on 10% sodium dodecyl sulfate -polyacrylamide gels (Bio-Rad; Hercules, CA). They were transferred to a nitrocellulose membrane and the

IL-6 ELISA H-1 and H-C cells (each 2  105 cells) were plated in 6-well plates. After 24-h incubation in serum-free medium, CM were harvested, and the IL-6

Fig. 1. Kindlin-2 was expressed in pancreatic cancer. (A-C, E, F) Immunohistochemical staining for kindlin-2 was performed in the resected pancreas cancer tissues (A-C), CP tissues (E), and normal pancreas (F). In normal pancreas, enlarged image of the pancreatic duct (arrow in panel E) is also presented. Nuclei were counterstained with hematoxylin. Original magnification: 200. (D) Double immunofluorescent staining for kindlin-2 (green) and a-SMA (red) was performed in the pancreatic cancer tissue. Nuclei were counterstained with 40 , 6-diamidino-2-phenylindole (blue). A merged photograph is also presented. Original magnification: 800. (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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concentrations in the CM of H-C and H-1 cells were determined by ELISA (Human IL6 ELISA Kit; Thermo Fisher scientific) according to the manufacturer's instruction.

Results Kindlin-2 was expressed in PDAC and activated PSCs

Indirect co-culture of pancreatic cancer cells and PSCs Pancreatic cancer cells (1  105 cells/well) were seeded into 6-well culture plates. The hPSC21-S/T cells (1  105 cells/culture insert) were seeded into culture inserts of 1.0 mm pore size. The next day, the culture inserts containing hPSC21-S/T cells were placed into 6-well plates containing pancreatic cancer cells, and incubation was continued up to 3 days in DMEM supplemented with 10% FBS, penicillin sodium and streptomycin sulfate.

Subcutaneous tumor model in nude mice Tumor formation in vivo was assessed by subcutaneously injecting SUIT-2 cells (1  106) alone or with PSCs (H-C or H-1 cells; 1  106) suspended in 100 ml of sterile PBS on the left side and right side of the back of male athymic nude mice (ICR-nu). For each group, two tumors per mouse developed in 6 mice (12 tumors in total). The tumor volume was measured every 7 days up to 28 days after the injection. The volume of a tumor was calculated using the formula V (mm3) ¼ (L X W2)/2 where L and W indicate the length and width of a tumor, respectively [25]. Immunohistochemical staining for a-SMA was performed as previously described [25]. Collagen accumulation was assessed by Sirius red staining, which preferentially labels collagen fibrils with red color [26].

We first examined the kindlin-2 expression in resected pancreatic tissues of patients undergoing operation for pancreatic cancer. Immunohistochemical staining showed that kindlin-2 was expressed in the PDAC and stroma (Fig. 1A). In some tissues, kindlin-2 expression was observed only in the stroma but not in the PDAC (Fig. 1B), or only in the PDAC but not in the stroma (Fig. 1C). In the stroma, kindlin-2 expression was strong in the peritumoral areas. Double immunofluorescent staining showed that kindlin-2 was co-localized with a-SMA, a marker of activated PSCs [8], indicating that activated PSCs expressed kindlin-2 (Fig. 1D). It has been established that activated PSCs play a pivotal role in the development of pancreatic fibrosis in CP [7,8]. We also examined the kindlin-2 expression in resected pancreatic tissues of patients undergoing operation for CP. Kindlin-2 expression was observed in spindle-shaped PSCs in the area of pancreatic fibrosis in CP (Fig. 1E). In normal pancreas, kindlin-2 expression was faintly observed in some pancreatic ductal cells, but was not obvious in pancreatic acinar cells (Fig. 1F).

Statistical analysis Data are shown as mean ± standard error (SE). Experiments were performed at least three times and similar results were obtained. The differences between two groups were analyzed by unpaired Student's t-test. The overall and reference-free survival time were calculated by the KaplaneMeier method and compared by a log-rank test. Statistical analyses were performed using the SPSS version 20.0 statistical analysis software (SPSS Inc., Chicago, IL). A P value less than 0.05 was considered statistically significant.

Stromal kindlin-2 expression was associated with shorter recurrence-free survival time We examined whether kindlin-2 expression was associated with the disease prognosis (overall or recurrence-free survival

Fig. 2. High stromal kindlin-2 expression was associated with shorter recurrence-free survival time. Kindlin-2 expression in PDAC and stroma, as assessed by immunohistochemical staining, was classified as high (>50%) or low (<50%). The overall survival (panels A and C) and recurrence-free survival (panels B and D) time of 79 patients stratified by high or low kindlin-2 expression in PDAC (panels A and B) and stroma (panels C and D) were calculated using the KaplaneMeier method and compared by a log-rank test.

N. Yoshida et al. / Cancer Letters 390 (2017) 103e114 Table 2 Relationship between stromal kindlin-2 expression and clinicopathological factors in patients with PDAC (n ¼ 79). Characteristics

High expression group

Low expression group

n ¼ 49 (62.0%)

n ¼ 30 (38.0%)

Age <65 25 (51.0%) 65 24 (49.0%) Sex Male 31 (63.3%) Female 18 (36.7%) Tumor site Head 37 (75.5%) Other 12 (24.5%) Tumor size (cm) 3.4 ± 0.15 T-UICC staging I/II 43 (87.8%) III/IV 6 (12.2%) Histologic grade 1/2 44 (89.8%) 3 5 (10.2%) pN category pN0 10 (29.4%) pN1 39 (79.6%) Lymphatic invasion ly1þ/2þ 28 (57.1%) ly3þ 21 (42.9%) Vascular invasion v1þ/2þ 23 (46.9%) v3þ 26 (53.1%) Perineural invasion ne1þ/2þ 19 (38.8%) ne3þ 30 (61.2%) Adjuvant chemotherapy No 5 (10.2%) Yes 44 (89.8%) Radiation therapy No 42 (85.7%) Yes 7 (14.3%)

P value

P ¼ 0.71 14 (46.7%) 16 (53.3%) P ¼ 0.76 20 (66.7%) 10 (33.3%) P ¼ 0.92 19 (63.3%) 11 (36.7%) 3.3 ± 0.19

P ¼ 0.70 P ¼ 0.36

24 (80.0%) 6 (20.0%) P ¼ 0.58 28 (93.3%) 2 (6.7%) P ¼ 0.34 9 (30.0%) 21 (70.0%) P ¼ 0.98 23 (76.7%) 7 (23.3%) P ¼ 0.41 17 (56.7%) 13 (43.3%) P ¼ 0.69 13 (43.3%) 17 (56.7%) P ¼ 0.67 4 (13.3%) 26 (86.7%) P ¼ 0.91 26 (86.7%) 4 (13.3%)

time) in 79 patients with pancreatic cancer who underwent R0 resection. Kindlin-2 expression was high in both the PDAC and the stroma in 35 cases, high in the PDAC but low in the stroma in 19 cases, low in the PDAC but high in 14 cases and low in both the PDAC and the stroma in 11 cases. When kindlin-2 expression was stratified by its location, high kindlin-2 expression in the PDAC was not associated with overall or recurrence-free survival time (Fig. 2).

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Table 2 shows the relationship between stromal kindlin-2 expression and the clinicopathological factors. On the other hand, high stromal kindlin-2 expression was associated with recurrence-free survival (P ¼ 0.025). Patients with high stromal kindlin-2 expression had shorter recurrence-free survival (725.2 þ 99.6 days) than those with low stromal kindlin-2 expression (1026.9 þ 112.7 days). The overall survival time tended to be shorter in patients with high stromal kindlin-2 expression than that in those with low stromal kindlin-2 expression, but the difference was not statistically significant (P ¼ 0.052). PSCs and pancreatic cancer cell lines expressed kindlin-2 in vitro We next examined the expression of kindlin-2 in pancreatic cancer cell lines, primary human PSCs, and immortalized human PSCs (hPSC21-S/T cells) in vitro. Quantitative real-time PCR (Fig. 3A) and Western blotting (Fig. 3B) showed that PSCs as well as pancreatic cancer cell lines, except for AsPC-1, expressed kindlin-2 both at the mRNA and protein levels. Kindlin-2 expression was high both in cancer-associated PSCs (hPSC1, hPSC2, and hPSC3) and CPassociated PSCs (hPSC4 and hPSC5). Overall, kindlin-2 expression appeared higher in PSCs than that in pancreatic cancer cells. Knockdown of kindlin-2 altered gene expression in PSCs To clarify the role of stromal kindlin-2 expression in the progression of pancreatic cancer, we established kindlin-2-kockdown PSC line by the introduction of siRNA against kindlin-2 to the human immortalized hPSC21-S/T cells. Two kindlin-2-knockdown cell lines were established and designated as H-1 and H-2 cells. Quantitative real-time PCR and Western blotting showed that kindlin-2 expression was decreased in the kindlin-2-knockdown H1 and H-2 cells compared to the control cell line (H-C cells) (Fig. 4A, 4B). Quantitative real-time PCR showed that kindlin-2 expression was decreased in H-1 cells to about 17% and in H-2 cells to about 24% of H-C cells. We conducted a microarray to identify the genes differentially expressed between H-1 and H-C cells. As shown in Fig. 4C, the gene expression profile was dynamically altered by kindlin-2 knockdown in PSCs. Based on the criteria described in Methods, the expression of 724 genes was upregulated and that of 495 genes was downregulated. The altered mRNAs were analyzed to identify the networks and pathways using the IPA. IPA revealed the most

Fig. 3. PSCs and pancreatic cancer cell lines expressed kindlin-2. (A) Total RNA was prepared from pancreatic cancer cell lines, pancreatic cancer-associated human primary PSCs (hPSC1, hPSC2, and hPSC3), CP-associated human primary PSCs (hPSC4 and hPSC5), and immortalized human PSCs (hPSC21-S/T cells). The mRNA level of kindlin-2 was determined by quantitative real-time PCR. The expression levels of kindlin-2 mRNAs were normalized by that of GAPDH. n ¼ 3 for each cell type. (B) Total cell lysates (approximately 100 mg) were prepared from each cell type and the levels of kindlin-2 and GAPDH were determined by Western blotting. Densitometry analysis was performed using Image J software. Kindlin-2 expression was normalized by the expression of GAPDH. Upper panel shows representative results of densitometry analysis.

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Fig. 4. Knockdown of kindlin-2 altered gene expression in PSCs. The kindlin-2-knockdown cell lines H-1 and H-2 were established by introducing the kindlin-2-siRNAexpressing vector, followed by selection with puromycin and limiting dilution, in hPSC21-S/T cells. The control cell line H-C was similarly established by introducing the nontargeting control siRNA-expressing vector. (A, C, D) Total RNAs were prepared from kindlin-2-knockdown hPSC21-S/T cells (H-1 and H-2 cells) and the control H-C cells. (B) Total cell lysates (approximately 100 mg) were prepared from H-C, H-1 cells, and H-2 cells. (A, D) The expression levels of kindlin-2, CXCL1, CXCL2, CCL2, IL-1b, IL-6, and IL-8 genes were determined by quantitative real-time PCR. The expression levels of each gene were normalized by that of GAPDH. (B) The levels of kindlin-2 and a-tubulin were determined by Western blotting. (C) Gene expression profiles were compared using Agilent's microarray and a heat map was generated. The color indicated the distance from the median of each row. ** indicates P < 0.01 vs. H-C cells (n ¼ 3 each). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

impacted biological processes including: cellular movement (539 molecules), cellular development (726 molecules), cell death and survival (692 molecules), and cellular growth and proliferation (817 molecules). IPA also identified TNF and IL-1b as top upstream regulators altered by kindlin-2 knockdown, suggesting a role of kindlin-2 in the maintenance of a pro-inflammatory phenotype in PSCs. Indeed, the expression of mRNAs for proinflammatory cytokines and chemokines including IL-8 (Z-score 7.1, ratio 0.03 vs. HC cells in microarray), CXCL1 (Z-score 6.5, ratio 0.07), CCL2 (Z-

score 6.5, ratio 0.13), CXCL2 (Z-score 6.0, ratio 0.09), IL-1b (Zscore 5.2, ratio 0.16), and IL-6 (Z-score 5.1, ratio 0.17) was downregulated by kindlin-2 knockdown. The suppression of these cytokines was confirmed by quantitative real-time PCR in H-1 and H-2 cells (Fig. 4D). The suppression of IL-6 expression by kindlin-2 knockdown was also confirmed by ELISA of CM. The IL-6 concentration in the CM of H-1 cells (376.7 þ 10.3 pg/ml) was significantly lower than that in the CM of H-C cells (976.0 þ 37.3 pg/ml) (P ¼ 0.0001; n ¼ 3 each).

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Fig. 5. Knockdown of kindlin-2 resulted in decreased proliferation and migration in immortalized PSCs. (A) H-C, H-1, or H-2 cells were seeded in 96-well plates in normal growth medium. Cell proliferation was assessed by BrdU incorporation ELISA, and optical density (OD) 370-OD492 of the samples is shown. (B) Cells were grown to confluence in a 12-well culture plate. Monolayers of H-C and H-1 cells were scratched with sterile 200-ml pipette tips. After 24 h, cell migration toward the scratched areas was observed. Orginal magnification: 40. (C, D) 1  104 H-C, H-1, or H-2 cells were seeded in serum-free medium in the upper chamber and migration during 18 h toward the lower chamber, which contained 10% FBS as a chemoattractant, was evaluated. Migrated cells were counted in 5 randomly chosen high power fields (HPFs). Original magnification: 100. Panel D summarizes the results. **:P < 0.01 vs. H-C cells (n ¼ 5 each).

Inhibition of kindlin-2 expression resulted in decreased proliferation and migration in PSCs Knockdown of kindlin-2 resulted in decreased proliferation in PSCs as assessed by BrdU incorporation assay (Fig. 5A). Knockdown of kindlin-2 decreased migration in PSCs as assessed by scratch assay (Fig. 5B) and two-chamber assay (Fig. 5C and D). PSC-induced EMT, proliferation and migration of pancreatic cancer cells were suppressed when kindlin-2 was knocked down in immortalized human PSCs Previous studies showed that PSCs induced proliferation, migration, epithelialemesenchymal transition (EMT) in pancreatic cancer cells [4,8,27]. We examined the impact of kindlin-2 knockdown in PSCs on PSC-induced alterations of cell functions in pancreatic cancer cells. Indirect co-culture with H-C cells induced a fibroblastic morphology in Panc-1 and SUIT-2 cells, but this effect was suppressed in the case of co-culture with the kindlin-2knockdown H-1 cells (Fig. 6A). The expression of mesenchymal markers snail and vimentin in pancreatic cancer cells was increased by the treatment with CM of H-C cells, but the effects were less evident in the case of CM of H-1 cells (Fig. 6B). Similarly, treatment with CM of H-C cells induced proliferation and migration of SUIT-2 cells, but this effect was less evident in the case of CM of H-1 cells (Fig. 6C and D).

PSC-induced proliferation and migration of pancreatic cancer cells were suppressed when kindlin-2 was knocked down in primary human PSCs We then examined the impact of kindlin-2 knockdown in primary human PSCs. For this purpose, primary human PSCs were transfected with the siRNA against kindlin-2 or the negative control siRNA. Quantitative real-time PCR showed that kindlin-2 expression was almost completely suppressed by the transfection of the siRNA against kindlin-2 (Fig. 7A). The expression of IL-1b and CCL2 genes was decreased by the transfection with the siRNA against kindlin-2 compared to the control siRNA (Fig. 7A). Treatment with CM of primary PSCs transfected with the control siRNA increased the proliferation and migration of SUIT-2 cells, but this effect was less evident in the case of CM of primary PSCs transfected with the siRNA against kindlin-2 (Fig. 7B and C). Inhibition of kindlin-2 in PSCs abolished tumor-supporting effects of PSCs in nude mice Finally, we examined the impact of kindlin-2 knockdown in PSCs on tumor-supporting effects of PSCs in vivo. SUIT-2 pancreatic cancer cells were injected alone, with the control HC cells, or with the kindlin2-knockdown H-1 cells. At 28 days, coinjection of the control PSCs (H-C cells) with SUIT-2 cells increased the size of subcutaneous tumors compared to SUIT-2

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cells alone (Fig. 8A and B) (P < 0.0001). But, this tumor-promoting effect was abolished when H-1 cells, but not H-C cells, were coinjected (P ¼ 0.0002 for SUIT-2þH-1 cells vs. SUIT-2þH-C cells; P ¼ 0.73 for SUIT-2 cells alone vs. SUIT-2 cellsþH-1 cells; n ¼ 12 tumors in 6 mice for each group). Compared to the injection of SUIT-2 cells alone, co-injection of the H-C cells increased the

collagen deposition and the number of activated PSCs in the subcutaneous tumors, as assessed by Sirius Red staining and immunohistochemical staining for a-SMA (Fig. 8C). But these effects were much less evident in the case of co-injection of H-1 cells. These results suggested that kindlin-2 promoted tumor growth in vivo.

Fig. 6. PSC-induced EMT, proliferation and migration of pancreatic cancer cells were suppressed when kindlin-2 was knocked down in immortalized PSCs. (A) Panc-1 or SUIT-2 panceatic cancer cells were monocultured or indirectly cocultured with H-C or H-1 cells in normal growth medium for 72 h. Morphological alternations were examined under phase-contrast microscopy. Original magnification: 100. (B) Panc-1 cells were left untreated (Control) or treated with conditioned media (CM) of H-C or H-1 cells for 72 h. Total RNAs were prepared, and the expression of mesenchymal markers snail and vimentin genes was determined by quantitative real-time PCR. The expression levels of each gene were normalized by that of GAPDH. The expression levels of each gene in Control were set as 1. (C) SUIT-2 cells were left untreated (Control) or treated with conditioned media (CM) of H-C or H-1 cells for 24 h. Cell proliferation was assessed by BrdU incorporation ELISA, and optical density (OD)370-OD492 of the samples is shown. (D) 1  104 SUIT-2 cells were seeded in serum-free medium in the upper chamber and migration during 18 h toward the lower chamber, which contained 10% FBS (Control) or CM of H-C or H-1 cells as a chemoattractant, was evaluated. Migrated cells were counted in 5 randomly chosen high power fields (HPFs). Original magnification: 100. * indicates P < 0.05 and ** indicates P < 0.01 vs. cells treated with CM of H-C (n ¼ 3 each in panel B, n ¼ 5 each in panels C and D). NS: not significant.

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Discussion The major findings of this study are as follows. First, kindlin-2 was expressed in pancreatic cancer cells and PSCs; second, stromal kindlin-2 expression was associated with shorter recurrencefree survival after R0 resection; third, knockdown of kindlin-2 resulted in decreased proliferation, migration and cytokine expression in PSCs; fourth, PSC-induced proliferation and migration were inhibited when kindlin-2 expression was knockdown in PSCs; lastly, the size of the subcutaneous tumor generated by the co-injection of SUIT-2 cells with PSCs was decreased when kindlin2 expression was knocked down in PSCs. Collectively, our results suggest that kindlin-2 in PSCs promotes the progression of pancreatic cancer. The aberrant expression of kindlin-2 has been reported in several types of cancer (reviewed in [14]). Kindlin-2 might exert tumor-promoting or tumor-inhibiting effects. For example, kindlin2 induced EMT and the invasion of breast cancer cells by activating Wnt signaling [28] and targeting miR-200b [29]. Kindlin-2 increased the invasiveness of prostate cancer cells through the up-regulation of matrix metalloproteinases mediated by NF-kB [30]. Kindlin-2 causes HOXB9 and E-cadherin repression in pancreatic cancer cells, as a downstream target of TGF-b signaling, which leads to increased proliferation, migration and invasion of pancreatic cancer cells [19]. In gastric cancer cells, kindlin-2

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contributes to the phosphorylation of b1 and b3 integrins and increased cellular migration [31]. In an inhibitory context, kindlin-2 inhibited the growth and migration of colon cancer cells through decreased phosphorylation of glycogen synthase kinase 3b and promoted the degradation of b-catenin, a principal provider of oncogenic signals in colorectal cancer [32]. Up-regulation of estrogen receptor a by kindlin-2 led to enhanced transcription of Ecadherin, resulting in decreased metastatic capacity [33]. Two previous studies have reported kindlin-2 expression in pancreatic cancer, but with conflicting results. Mahawithitwong et al. [18] reported that kindlin-2 was highly expressed in the stroma but not in the PDAC, whereas Zhan et al. [19] reported high expression of kindlin-2 in PDAC cells. Immunohistochemical staining in this study showed that there were four patterns of kindlin-2 in pancreatic cancer: high and low in the PDAC and stroma. Thirty five out of 79 (44%) cases showed high kindlin-2 expression in both the PDAC and stroma. Such heterogeneity might explain the conflicting results of the previous studies. In addition to pancreatic cancer tissues, stromal kindlin-2 expression was observed in CP tissues. Knockdown of kindlin-2 decreased the proliferation, migration, and cytokine expression in PSCs, suggesting a role of kindlin-2 in pancreatic fibrosis in general. This is consistent with previous studies in other organs showing a role of kindlin-2 in fibrosis [34,35]. It has been reported that kindlin-2 mediates the interaction between TGF-b type I receptor and

Fig. 7. PSC-induced proliferation and migration of pancreatic cancer cells were suppressed when kindlin-2 was knocked down in primary PSCs. (A) Primary human PSCs were transfected the siRNA against kindlin-2 or the control siRNA. The mRNA levels of kindlin-2, IL-1b, and CCL2 were determined by quantitative real-time PCR. The expression levels of each gene were normalized by that of GAPDH. The expression levels of each gene in the cells transfected with the control siRNA were set as 1. **: P < 0.01 vs. the cells transfected with the control siRNA (n ¼ 3 each). (B, C) Conditioned media (CM) were prepared from primary PSCs transfected with the siRNA against kindlin-2 or the control siRNA. (B) SUIT-2 cells were left untreated (Control) or treated with the CM for 24 h. Cell proliferation was assessed by BrdU incorporation ELISA, and optical density (OD) 370-OD492 of the samples is shown. (C) 1  104 SUIT-2 cells were seeded in serum-free medium in the upper chamber and migration during 18 h toward the lower chamber, which contained normal growth medium (Control) or CM as a chemoattractant, was evaluated. Migrated cells were counted in 5 randomly chosen high power fields (HPFs). Original magnification: 100. * indicates P < 0.05 and ** indicates P < 0.01. NS: not significant.

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Smad3, leading to increased Smad3 activation in kidney tubular epithelial cells [34]. Suppression of this interaction by kindlin-2 knockdown attenuated renal fibrosis, indicating a fibrosispromoting role of kindlin-2. In skin, kindlin-2 became highly expressed in myofibroblasts upon activation in healing wounds [35]. In this study, stromal kindlin-2 expression was significantly associated with recurrence-free survival time after R0 resection. This is in agreement with a previous study [18] showing that stromal kindlin-2 expression was an independent risk factor for poor prognosis in patients with PDAC after R0 resection. Further prospective studies are warranted to clarify whether stromal kindlin-2 expression would be useful to identify patients at high risk for recurrence. On the other hand, the mechanism underlying this finding remains to be clarified. One possible mechanism might involve cancer stem cells. Cancer stem cells within the entire cancer tissue harbor highly tumorigenic and chemo-resistant phenotypes, which lead to the recurrence after surgery or re-growth of the tumor [36]. Of note, it has been reported that PSCs might act as a part of the cancer stem cell niche [23]. PSCs enhanced the spheroidforming ability of cancer cells and induced the expression of

cancer stem cell-related genes ABCG2, Nestin and LIN28. In addition, co-injection of PSCs enhanced tumorigenicity of pancreatic cancer cells in vivo. IPA revealed the most impacted biological processes by the kindlin-2 knockdown included cellular movement, cellular growth, and proliferation. Indeed, knockdown of kindlin-2 resulted in decreased proliferation and migration in PSCs. This finding is not unexpected, because b1-integrin signaling is essential for cellular proliferation and migration in PSCs [37]. Liu et al. [38] reported that kindlin-2 interacts with Src-focal adhesion kinase pathway, which is involved in cellular migration in PSCs [39]. In addition, several studies have shown that kindlin-2 interacts with several signaling pathways, independently of integrins, such as Wnt/b-catenin [28], TGF-b/Smad [34] and TGF-b1-HOXB9-paxillin signaling pathway [19]. Further studies are required to clarify whether kindlin-2 regulate these signaling pathways in PSCs. IPA also identified TNF and IL-1b as top upstream regulators altered by kindlin-2 knockdown, suggesting a role of kindlin-2 in the maintenance of a proinflammatory phenotype in PSCs. In agreement with the results of IPA, knockdown of kindlin-2 decreased migration, proliferation and IL-6 expression in PSCs. Decreased IL-6 expression by kindlin-2

Fig. 8. Knockdown of kindlin-2 in PSCs abolished tumor-supporting effects of PSCs. SUIT-2 cells (1  106 cells) were subcutaneously injected on the left side and right side of the back of nude mice alone or with the control H-C or kindlin-2-knockdown H-1 cells (each 1  106 cells). For each group, two tumors per mouse developed in 6 mice (12 tumors in total). (A) Tumor sizes were measured every 7 days. At 28 days after the injection, the mice were sacrificed and subcutaneous tumors were resected. (B) Appearance of the resected tumors. (C) Sirius Red staining and immunohistochemical staining of a-SMA were performed on the resected tumor sections. Nuclei were counterstained with hematoxylin. Original magnification: 200.

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knockdown suggests a role of b-integrins-kindlin-2 pathways in optimal IL-6 production in PSCs. Indeed, a previous study showed that blockade of avb3 and a5b1 integrins inhibited fibrinogeninduced IL-6 production in PSCs [22]. Very recently, we have shown that IL-6 is a regulator of PSC-induced EMT in pancreatic cancer cells [10]. Decreased IL-6 production by kindlin-2 in PSCs would explain the suppression of PSC-induced fibroblast-like morphological changes and expression of mesenchymal markers in pancreatic cancer cells. This study revealed a novel mechanism responsible for the tumor-promoting effects of kindlin-2 in pancreatic cancer: a regulatory role of kindlin-2 in PSCs in the interactions between PSCs and pancreatic cancer cells. Because previous studies have shown that PSCs promote the progression of pancreatic cancer [4e10], PSCs have attracted the attention of researchers as a novel therapeutic target for the treatment of pancreatic cancer. Importantly, recent studies have presented paradigm-shifting results, suggesting that PSCs might have inhibitory effects on the progression of pancreatic cancer [40,41]. Simple removal of stromal cells from pancreatic cancer exacerbated cancer cell dissemination, leading to shorter survival in mice. Reprograming, rather than removal, of the stroma might be an option for the treatment of pancreatic cancer [42]. Previous studies have shown that PSCs expressed a wide range of integrins and that the cell functions of PSCs were regulated by integrin-mediated signaling. For example, Gao and Brigstock [43] reported that connective tissue growth factor increased proliferation and collagen synthesis in rat PSCs through a5b1 integrin receptor. Fibrinogen induced IL-6 and IL-8 production through avb3 and a5b1 integrins [22]. Chen et al. [37] reported that human fetal PSCs expressed a3b1 and a5b1 integrins, and that maintaining stellate cell functions and proliferation required the interaction between b1 integrin and type 1 collagen matrix. Mice with b1 integrin deficiency under control of the type I collagen promoter directly affected the PSC function and survival [44]. These findings suggest that integrin signaling might be a novel therapeutic target in pancreatic cancer through altering the stellate cell phenotype. Importantly, it has been well recognized that integrins act as cellsurface receptors for extracellular matrix and matricellular proteins, and regulate a variety of cell functions in PDAC cells [45,46]. It is reasonable to assume that broad inhibition of integrin signaling might be more efficient than that of the respective integrins. From this viewpoint, kindlin-2 is an attractive target because kindlin-2 is an essential element in integrin activation [15]. As described above, kindlin-2 is expressed in both pancreatic cancer cells and PSCs. Zhan et al. [18] reported that stable knockdown of kindlin-2 inhibited the growth, migration, invasion, and EMT in PDAC cells in vitro. In addition, we have recently found that the growth of xenograft tumors was inhibited if kindlin-2 was knocked down in SUIT-2 cells (Masamune et al. unpublished observations). Therefore, kindlin-2 inhibitors might target both PSCs and PDAC cells. Very recently, 3-arylquinoline and 3-aryl-2-quinolone derivatives, chemically close to flavonoids, have been identified as a new class of integrin antagonist that interferes with the interaction between b3 integrin cytoplasmic tail and kindlin-2 [47]. It would be of interest to determine whether these antagonists could be useful for the treatment of pancreatic cancer. Financial supports This study was supported in part by Grant-in-Aid from the Japan Society for the Promotion of Science (26293171, 26461029, 15H04804), the Pancreas Research Foundation of Japan (to T. Takikawa), the Mitsui Life Social Welfare Foundation (to A. Masamune), and the Smoking Research Foundation (to A. Masamune).

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The funding agencies had no role in the design of the study or preparation of this manuscript, and will have no influence on the data collection, analysis, and interpretation or manuscript publication.

Conflicts of interest None declared.

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