LASP1 is an actin-binding protein associated with actin assembly dynamics in cancer cells. Here, we report that LASP1 is overexpressed in pancreatic ductal adenocarcinoma (PDAC) where it promotes invasion and metastasis. We found that LASP1 overexpression in PDAC cells was mediated by HIF1α through direct binding to a hypoxia response element in the LASP1 promoter. HIF1α stimulated LASP1 expression in PDAC cells in vitro and mouse tumor xenografts in vivo. Clinically, LASP1 overexpression in PDAC patient specimens was associated significantly with lymph node metastasis and overall survival. Overall, our results defined LASP1 as a direct target gene for HIF1α upregulation that is critical for metastatic progression of PDAC. Cancer Res; 75(1); 111–9. ©2014 AACR.

Despite recent advances in the diagnosis and therapeutic modalities, pancreatic ductal adenocarcinoma (PDAC) remains the digestive tract tumor with the poorest prognosis (1). PDAC is the fourth highest cause of cancer-related deaths in the United States, with a 5-year survival rate lower than 5% (2). The aggressive course of PDAC progression is mostly due to delayed diagnosis, resistance to conventional chemotherapies, and development of metastasis (3). The underlying mechanisms that trigger PDAC invasion and metastasis remained largely unknown, although it is believed that the hypoxic microenvironment and hypoxia-induced factors are critical drivers of PDAC metastasis and progression (4).

LIM and SH3 protein 1 (LASP-1) is an actin-binding protein containing an N-terminal LIM domain and two actin-binding domains in the core of LASP-1 protein (5). LASP-1 was first identified from a cDNA library of metastatic axillary lymph nodes of patients with breast cancer, and the gene was mapped to human chromosome 17q21 (6, 7). LASP-1 interacts with the actin cytoskeleton at the site of cell membrane extensions, but not along actin stress fibers (5, 8–10). The SH3 domain at the C-terminus is involved in interactions with zyxin, pallidin, lipoma-preferred partner, and vasodilator-stimulated phosphoprotein (9, 11). LASP-1 reportedly localizes within multiple sites of dynamic actin assembly, such as focal contacts, focal adhesions, lamellipodia, membrane ruffles, and pseudopodia, and is involved in cell proliferation and migration (12). It was previously reported that LASP-1 overexpression induced cell proliferation and migration in human breast cancer, ovarian cancer, colorectal cancer, malignant childhood medulloblastoma, and hepatocellular carcinoma (5, 13–16). However, the role of LASP-1 in PDAC progression has not been examined.

In this study, we aim to investigate the role of LASP-1 in PDAC progression. Our data showed that LASP-1 was overexpressed in PDAC, and LASP-1 overexpression promoted PDAC cell migration and invasion in vitro and metastasis in xenograft mouse models. LASP-1 overexpression in PDAC is mediated by HIF1α, which directly binds to and transactivates the LASP-1 promoter. Our findings indicate that LASP-1 is a novel direct HIF1α target gene that promotes PDAC metastasis and progression.

Cell culture and hypoxic treatment

Human PDAC cell lines, CFPAC-1, BxPC-3, and Panc-1 were obtained from the Committee of Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China) and MIA-PaCa-2 was obtained from the ATCC. All the cell lines were obtained in 2013, and recently authenticated in August 2014 through the short tandem repeat analysis method. These cells were grown at 37°C in a humidified atmosphere of 95% air and 5% CO2 using DMEM with 10% FBS. For hypoxic treatment, cells were placed in a modulator incubator (Thermo Electron Co.) in an atmosphere consisting of 93.5% N2, 5%CO2, and 1.5% O2.

Western blot analysis

Whole-cell extracts were prepared by lysing cells with RIPA lysis buffer supplemented with a proteinase inhibitor cocktail (Sigma). Protein concentrations were quantified using Pierce Protein Assay Kit (Pierce). Protein lysates (20 μg) were separated by SDS-PAGE, and target proteins were detected by Western blot analysis with antibodies against HIF1α (1:1,000), LASP-1 (1:2,000), and β-actin (1:1,000; Supplementary Table S1). Specific proteins were visualized using an Enhanced Chemiluminescence Detection Reagent (Pierce).

Reverse transcription PCR

Total RNA was isolated from transfected cells with TRIzol Reagent (Invitrogen) and used for first-strand cDNA synthesis using the First-Strand Synthesis System for reverse transcription PCR (RT-PCR, Takara). Each sample was processed in triplicate, and β-actin was used as loading control. Each experiment was repeated independently at least three times. PCR primers used are indicated in Supplementary Table S1.

Immunofluorescence

To assess LASP-1 and F-actin distribution, human PDAC cells were seeded onto glass slides for different treatments. The cells were then washed once with PBS and fixed with 4% paraformaldehyde in PBS for 15 minutes, permeabilized with 0.1% Triton X-100 in PBS for 30 minutes at room temperature, and blocked for 1 hour with 3% BSA in PBS. Then cells were stained with anti-LASP-1 antibody (1:200 dilution, overnight at 4°C). F-actin was stained with phalloidin-FITC (Beyotime Biotechnology). Cells were mounted with DAPI Fluoromount-G media with DAPI nuclear stain (Southern Biotech). Slides were viewed with Olympus confocal microscopy.

Chromatin immunoprecipitation assay

Chromatin immunoprecipitation assay was performed using a commercial kit (Upstate Biotechnology) according to the manufacturer's instructions. Primers flanking the hypoxia response elements (HRE) of the VEGF promoter were used as a positive control (17, 18). The PCR primers are indicated in Supplementary Table S1.

siRNA duplexes, plasmid constructs, transient transfection, and stable transfection in pancreatic cancer cells and luciferase assay

siRNAs against LASP-1 and HIF1α were designed and synthesized from GenePharma (Supplementary Table S1). The human LASP-1 cDNA was cloned into the pcDNA3.1 plasmid expression vector. The pcDNA3.1-HIF1α plasmids were prepared as previously described (17, 18).

LASP-1 overexpression in Panc-1 cells, lentivirus-mediated plasmid was done using the pLV-cDNA system (Biosettia) following the manufacturer's instructions. Lentivirus encoding DNA were packaged as previously described (19). After transfection, the medium containing lentivirus was collected, filtered, and transferred onto Panc-1 cells. Infected cells were selected with puromycin (1 μg/mL) for 7 days.

Genomic DNA fragments of the human LASP-1 gene, spanning from +1 to −2,000 relative to the transcription initiation sites were generated by PCR and inserted into pGL3-Basic vectors (denoted as pGL3-LASP-1). All constructs were sequenced to confirm their identity. Luciferase activity was measured using the Dual-Luciferase Reporter Assay System (Promega) as previously described (17, 18). For transfection, cells were plated at a density of 5 × 105 cells/well in 6-well plates with serum-containing medium. When the cells were 80% confluent, the siRNA duplexes or overexpression plasmids were transfected into cells using Lipofectamine 2000 (Invitrogen) for 48 hours. The cells were collected for cell migration and invasion analysis, Western blot analysis, RT-PCR, and immunofluorescence, etc.

Wound healing and cell migration assay

A wound-healing assay was performed according to published protocol (20). Invasion assays were performed with 8.0-μm pore inserts in a 24-well Transwell. For this assay, 1 × 105 cells were isolated and added to the top chamber of a Transwell with DMEM. The invasion assay was performed using 1/6 diluted Matrigel (BD Biosciences) coated filters. DMEM with 10% FBS was added to the bottom chamber and the cells were allowed to incubate for 24 hours. Cells that had migrated to the bottom of the filter were stained with a three-step stain set (Thermo Scientific). All experiments were repeated independently at least three times.

Animal studies and measurement of metastasis in orthotopic pancreatic cancer mouse model

Female 4-week-old nude nu/nu mice were maintained in a barrier facility on high-efficiency particulate air (HEPA)-filtered racks. All animal studies were conducted under an approved protocol in accordance with the principles and procedures outlined in the NIH Guide for the Care and Use of Laboratory Animals. Digoxin and saline for injection were obtained from Tianjin Medical University Cancer Institute and Hospital (Shanghai, China). Cells were harvested by trypsinization, washed in PBS, resuspended at 107cells/mL in a 1:1 solution of PBS/Matrigel, and injected subcutaneously into the right flank of 16 nude nu/nu mice. Primary tumors were measured in three dimensions (a, b, c), and volume was calculated as abc × 0.52 (21). Primary tumors were harvested from the flank of nude nu/nu mice. Part of the tumor was fixated by formalin and embedded using paraffin, and the rest of the tumor was used for protein extraction.

For orthotopic tumor cell injection, 18 nude nu/nu mice were divided into three groups (Panc-1/pLV Vector + saline, Panc-1/pLV Vector + digoxin, and Panc-1/pLV LASP-1 + digoxin, respectively, 6 mice each). A total of 2.0 × 106 cells were injected into the carefully exposed pancreas of nude mice. The pancreas was then returned to the peritoneal cavity; the abdominal wall and the skin was closed with skin clips. Six weeks later, the number of visible metastatic lesions in the gut, mesentery, and liver was determined (22).

Immunohistochemistry

With approval from the Ethics Committee, PDAC samples were obtained from 91 patients (aged 36–83 years) undergoing surgical resection with histologic diagnosis of PDAC at the Tianjin Cancer Institute and Hospital (Shanghai, China). Immunohistochemistry for HIF1α and LASP-1 of PDAC patient tissues was performed using a DAB Substrate Kit (Maxin). Immunoreactivity was semiquantitatively scored according to the estimated percentage of positive tumor cells as previously described (18). Staining intensity was scored 0 (negative), 1 (low), 2 (medium), and 3 (high). Staining extent was scored 0 (0% stained), 1 (1%–25% stained), 2 (26%–50% stained), and 3 (51%–100% stained). The final score was determined by multiplying the intensity scores with staining extent and ranged from 0 to 9. Final scores (intensity score × percentage score) less than 2 were considered as negative staining (−), 2–3 were low staining (+), 4–6 were medium staining (++), and >6 were high staining (+++).

Statistical analysis

The Student t test for paired data was used to compare mean values. ANOVA is used to analyze the data of two groups with continuous variables. Nonparametric data were analyzed with the Mann–Whitney U test. The categorical data were analyzed by either Fisher exact or χ2 method. Each experiment was conducted independently at least three times, and values were presented as mean ± SD, unless otherwise stated. Analyses were performed using SPSS18.0 statistical analysis software.

LASP-1 overexpression promoted PDAC cell migration and invasion

To determine the role of LASP-1 in PDAC progression, we compared its expression levels in PDAC specimens and paired adjacent normal pancreatic tissues from PDAC patients. Despite interindividual variations, LASP-1 protein (Fig. 1A) and mRNA (Fig. 1B) levels were found to be evidently upregulated in PDAC samples when compared with adjacent normal pancreatic tissues, suggesting that LASP-1 was activated at transcriptional levels during PDAC progression. Furthermore, LASP-1 signal measurement by immunohistochemistry was detected in most (87.9%) of PDAC tissues (Fig. 1C). Intriguingly, in pancreas tumors other than malignant PDAC, such as serous cystadenoma and neuroendocrine tumors, LASP-1 expression was negative (Fig. 1C).

Figure 1.

LASP-1 is overexpressed in the specimens of PDAC. A, Western blot analysis (left) of LASP-1 levels in ten total paired human PDAC tumorous and matched adjacent nontumorous tissues. LASP-1 protein expression levels were normalized by those of β-actin. The chart shows LASP-1 protein expression levels in PDAC specimens versus paired adjacent normal pancreatic tissues. N, normal; T, tumor; **, P < 0.01 (right). B, RT-PCR analysis of LASP-1 levels in seven total paired human PDAC tumorous and matched adjacent nontumorous tissues (left). LASP-1 mRNA expression levels were normalized by β-actin. The chart shows LASP-1 mRNA expression levels in PDAC specimens versus paired adjacent normal pancreatic tissues. N, normal; T, tumor; *, P < 0.05 (right). C, expression analysis of LASP-1 protein in PDAC, serous cystadenoma, and neuroendocrine tumor tissues by immunohistochemistry (magnification, ×200 or ×400).

Figure 1.

LASP-1 is overexpressed in the specimens of PDAC. A, Western blot analysis (left) of LASP-1 levels in ten total paired human PDAC tumorous and matched adjacent nontumorous tissues. LASP-1 protein expression levels were normalized by those of β-actin. The chart shows LASP-1 protein expression levels in PDAC specimens versus paired adjacent normal pancreatic tissues. N, normal; T, tumor; **, P < 0.01 (right). B, RT-PCR analysis of LASP-1 levels in seven total paired human PDAC tumorous and matched adjacent nontumorous tissues (left). LASP-1 mRNA expression levels were normalized by β-actin. The chart shows LASP-1 mRNA expression levels in PDAC specimens versus paired adjacent normal pancreatic tissues. N, normal; T, tumor; *, P < 0.05 (right). C, expression analysis of LASP-1 protein in PDAC, serous cystadenoma, and neuroendocrine tumor tissues by immunohistochemistry (magnification, ×200 or ×400).

Close modal

Next, we set out to examine the effects of LASP-1 overexpression on PDAC cell migration and invasion. To investigate the role of LASP-1 in the aggressive phenotypes of PDAC cells in vitro, we used siRNA transfection to knock down LASP-1 expression in two PDAC cell lines with high endogenous LASP-1 levels (CFPAC-1 and MIA-PaCa-2; Fig. 2A). Out of the three pairs of siRNA, siRNA #3 most efficiently knocked down LASP-1 expression by more than 70% (Fig. 2B and C), and was used in the subsequent functional studies. Cell migration and invasion analysis using Transwell assay suggested that LASP-1 depletion in CFPAC-1 and MIA-PaCa-2 cells evidently reduced cell migration and invasion (Fig. 2D). In the wound-healing assays, the migratory activity of CFPAC-1 and MIA-PaCa-2 cells was also inhibited by LASP-1 silencing (Fig. 2E). Immunofluorescence analysis confirmed regional colocalization between LASP-1 and F-actin (Fig. 2F). Interestingly, compared with CFPAC-1/siNC and MIA-PaCa-2/siNC cells, the morphology of CFPAC-1/siLASP-1 and MIA-PaCa-2/siLASP-1 cells lacked thin and long pseudopods (Fig. 2F). These data indicated that LASP-1 was critical for the migration and invasion of PDAC cells.

Figure 2.

LASP-1 promotes PDAC cell migration and invasion. A, the basic expression of LASP-1 protein in four human PDAC cells was assessed by Western blot analysis. B and C, LASP-1 protein expression in CFPAC-1 (B) and MIA-PaCa2 (C) cells transfected with negative control siRNA (siNC) and LASP-1 siRNA (siLASP-1#1–3; 50 nmol/L) for 48 hours was determined by Western blot analysis. β-Actin was used as a loading control. D, comparison of migration and invasion potential of CFPAC-1 (top) and MIA-PaCa2 (bottom) cells transfected with negative control (NC) siRNA and LASP-1 siRNA#3 (50 nmol/L) for 48 hours using Boyden chambers. The experiments were performed independently three times. *, P < 0.05 versus control. (magnification, ×200). E, wound-healing assays comparing the motility of CFPAC-1 (left) and MIA-PaCa2 (right) cells transfected with negative control siRNA and LASP-1 siRNA#3 (50 nmol/L) for 48 hours (magnification, ×100). F, confocal images of CFPAC-1 (left) and MIA-PaCa2 (right) cells transfected with NC siRNA and LASP-1 siRNA#3 (50 nmol/L) stained for LASP-1 (red), F-actin (green), and 4′,6-diamidino-2-phenylindole (DAPI, blue; magnification, ×600). Colocalization is indicated by the merged images showing yellow immunofluorescence. G, comparison of the migration and invasion potential of BxPC-3 (top) and Panc-1 (bottom) cells transfected with pcDNA3.1 and pcDNA3.1-LASP1 plasmids (2 μg) for 48 hours using Boyden chambers. The experiments were performed independently three times. *, P < 0.05 versus control (magnification, ×200). H, wound-healing assays comparing the migration of BxPC-3 (left) and Panc-1 (right) cells transfected with pcDNA3.1 and pcDNA3.1-LASP1 plasmids (2 μg) for 48 hours (magnification, ×100). I, confocal images of BxPC-3 (left) and Panc-1 (right) cells transfected with pcDNA3.1 and pcDNA3.1-LASP1 plasmids (2 μg) stained for F-actin (red) and DAPI (blue; magnification, ×600). Colocalization was indicated by the merged images showing yellow immunofluorescence.

Figure 2.

LASP-1 promotes PDAC cell migration and invasion. A, the basic expression of LASP-1 protein in four human PDAC cells was assessed by Western blot analysis. B and C, LASP-1 protein expression in CFPAC-1 (B) and MIA-PaCa2 (C) cells transfected with negative control siRNA (siNC) and LASP-1 siRNA (siLASP-1#1–3; 50 nmol/L) for 48 hours was determined by Western blot analysis. β-Actin was used as a loading control. D, comparison of migration and invasion potential of CFPAC-1 (top) and MIA-PaCa2 (bottom) cells transfected with negative control (NC) siRNA and LASP-1 siRNA#3 (50 nmol/L) for 48 hours using Boyden chambers. The experiments were performed independently three times. *, P < 0.05 versus control. (magnification, ×200). E, wound-healing assays comparing the motility of CFPAC-1 (left) and MIA-PaCa2 (right) cells transfected with negative control siRNA and LASP-1 siRNA#3 (50 nmol/L) for 48 hours (magnification, ×100). F, confocal images of CFPAC-1 (left) and MIA-PaCa2 (right) cells transfected with NC siRNA and LASP-1 siRNA#3 (50 nmol/L) stained for LASP-1 (red), F-actin (green), and 4′,6-diamidino-2-phenylindole (DAPI, blue; magnification, ×600). Colocalization is indicated by the merged images showing yellow immunofluorescence. G, comparison of the migration and invasion potential of BxPC-3 (top) and Panc-1 (bottom) cells transfected with pcDNA3.1 and pcDNA3.1-LASP1 plasmids (2 μg) for 48 hours using Boyden chambers. The experiments were performed independently three times. *, P < 0.05 versus control (magnification, ×200). H, wound-healing assays comparing the migration of BxPC-3 (left) and Panc-1 (right) cells transfected with pcDNA3.1 and pcDNA3.1-LASP1 plasmids (2 μg) for 48 hours (magnification, ×100). I, confocal images of BxPC-3 (left) and Panc-1 (right) cells transfected with pcDNA3.1 and pcDNA3.1-LASP1 plasmids (2 μg) stained for F-actin (red) and DAPI (blue; magnification, ×600). Colocalization was indicated by the merged images showing yellow immunofluorescence.

Close modal

To determine whether LASP-1 overexpression is sufficient to promote PDAC cell migration, LASP-1 was ectopically expressed in two PDAC cell lines with low endogenous LASP-1 levels (BxPC-3 and Panc-1; Supplementary Fig. S1). Cell migration and invasion analysis by Transwell assay suggested that LASP-1 upregulation in BxPC-3 and Panc-1 cells evidently increased cell migration and invasion (Fig. 2G). Wound-healing assays showed that the migratory activity of the BxPC-3 and Panc-1 cells was enhanced by LASP-1 overexpression when compared with the control cells (Fig. 2H). Importantly, BxPC-3/pcDNA3.1-LASP1 and Panc-1/pcDNA3.1-LASP1 displayed some pseudopods extending from cell bodies (Fig. 2I). These results showed that ectopic overexpression of LASP-1 was sufficient to promote PDAC cell migration and invasion.

HIF1α directly regulated the expression of LASP-1 through binding to the HRE in the LASP-1 gene promoter

Our previous study indicated HIF1 as a critical transcriptional factor in pancreatic cancer cell migration (18, 23). To determine whether HIF1 regulates transcription of LASP-1 in pancreatic cancer cells, we used specific siRNAs targeting HIF1α to effectively reduce HIF1α expression (Supplementary Fig. S2). Knockdown of HIF1α expression decreased LASP-1 mRNA (Fig. 3A, left) and protein (Fig. 3A, right) expression (P < 0.05). Moreover, when HIF1α was overexpressed in Panc-1 cells, LASP-1 mRNA (Fig. 3B, left) and protein (Fig. 3B, right) expression markedly increased (P < 0.05). Taken together, these results suggest that HIF1α plays a critical role in LASP-1 expression.

Figure 3.

LASP-1 is a novel HIF1α target gene critical for HIF1α-induced cell migration. A, CFPAC-1 cells were transfected with siHIF1α #3 (50 nmol/L) for 48 hours and assessed HIF1α and LASP-1 expression by quantitative RT-PCR (qRT-PCR; left) and Western blot (right) analysis. The experiments were performed independently three times. *, P < 0.05 versus control. B, Panc-1 cells were transfected with pcDNA3.1-HIF1α plasmids (2 μg) for 48 hours and HIF1α and LASP-1 expression was accessed by qRT-PCR (left) and Western blot (right) analysis. The experiments were performed independently three times. *, P < 0.05 versus control. C, HIF1α and LASP-1 expression was determined by Western blot analysis on four human PDAC cells cultured under normoxia (21% O2) and hypoxia (1.5% O2) for 12 hours. N, normoxia; H, hypoxia. D, the DNA sequence of the LASP-1 promoter. Four HRE sites are located at the different sites. E, chromatin immunoprecipitation analysis HIF1 binding to LASP-1 promoter in Panc-1 cells (top). The PCR products of VEGF promoter were used as positive control. H, N, PC, and NC indicate hypoxia, normoxia, positive control, and negative control, respectively. Bottom, the comparison of gray value between normoxia and hypoxia in HRE1; **, P < 0.01. The experiments were performed independently three times. F, luciferase analysis in Panc-1 cells. Panc-1 cells overexpressing HIF1α expression plasmids (pcDNA-HIF1α) or control vector (pcDNA3.1) were transfected with pGL3-LASP-1-promoter, pGL3-LASP-1-promoter mutation, pGL3-VEGF-promoter, or pGL3-empty vector. After transfection for 48 hours, cells were subjected to dual luciferase analysis. Results were expressed as a fold induction relative to the cells transfected with the control vector (pcDNA3.1) after normalization to Renilla activity. The experiments were performed independently three times.

Figure 3.

LASP-1 is a novel HIF1α target gene critical for HIF1α-induced cell migration. A, CFPAC-1 cells were transfected with siHIF1α #3 (50 nmol/L) for 48 hours and assessed HIF1α and LASP-1 expression by quantitative RT-PCR (qRT-PCR; left) and Western blot (right) analysis. The experiments were performed independently three times. *, P < 0.05 versus control. B, Panc-1 cells were transfected with pcDNA3.1-HIF1α plasmids (2 μg) for 48 hours and HIF1α and LASP-1 expression was accessed by qRT-PCR (left) and Western blot (right) analysis. The experiments were performed independently three times. *, P < 0.05 versus control. C, HIF1α and LASP-1 expression was determined by Western blot analysis on four human PDAC cells cultured under normoxia (21% O2) and hypoxia (1.5% O2) for 12 hours. N, normoxia; H, hypoxia. D, the DNA sequence of the LASP-1 promoter. Four HRE sites are located at the different sites. E, chromatin immunoprecipitation analysis HIF1 binding to LASP-1 promoter in Panc-1 cells (top). The PCR products of VEGF promoter were used as positive control. H, N, PC, and NC indicate hypoxia, normoxia, positive control, and negative control, respectively. Bottom, the comparison of gray value between normoxia and hypoxia in HRE1; **, P < 0.01. The experiments were performed independently three times. F, luciferase analysis in Panc-1 cells. Panc-1 cells overexpressing HIF1α expression plasmids (pcDNA-HIF1α) or control vector (pcDNA3.1) were transfected with pGL3-LASP-1-promoter, pGL3-LASP-1-promoter mutation, pGL3-VEGF-promoter, or pGL3-empty vector. After transfection for 48 hours, cells were subjected to dual luciferase analysis. Results were expressed as a fold induction relative to the cells transfected with the control vector (pcDNA3.1) after normalization to Renilla activity. The experiments were performed independently three times.

Close modal

To determine whether hypoxia may induce LASP-1 overexpression in PDAC cells, four PDAC cell lines were cultured under normoxia (21% O2) and hypoxia (1.5% O2) for 12 hours and the LASP-1 expression levels were determined by Western blot analysis. As shown in Fig. 3C, LASP-1 expression increased about 3.85-fold after hypoxic treatment when compared with normoxia-cultured cells, suggesting that hypoxic PDAC microenvironment might be responsible for LASP-1 overexpression.

To understand the molecular mechanism underlying LASP-1 overexpression in PDAC, we surveyed the promoter region of human LASP-1 gene and identified four HRE (Fig. 3D).

To investigate whether HIF1α directly binds to LASP-1 promoter, chromatin immunoprecipitation assay was performed in Panc-1 cells at 1.5% O2 or 21% O2. In chromatin fractions pulled down by an anti-HIF1α antibody, only the HRE of LASP-1 promoter located at −1,005 to −1,001 was detected (Fig. 3E, top). The fragment immunoprecipitated by anti-HIF1α antibody significantly increased (Fig. 3E, bottom; P < 0.01) under hypoxia, suggesting that hypoxia promoted the binding of HIFα to LASP-1 promoter.

To determine whether the binding of HIF1α activates LASP-1 promoter, we constructed a full-length LASP-1 luciferase promoter vector (containing HREs, −1,005 to 1,001) and cotransfected this reporter construct with or without HIF1α cDNA into Panc-1 cells. Luciferase analysis showed that HIF1α overexpression (pcDNA-HIF1α) significantly increased LASP-1 promoter activity in Panc-1 cells (∼2.9-fold, P < 0.05; Fig. 3F). To determine whether the HRE1 site is required for HIF1α to transactivate LASP-1 promoter, this HIF1α binding site was mutated from ACGTG to ACATG. As shown in Fig. 3F, the mutation of HRE1 almost abolished the transactivation of LASP-1 promoter by HIF1α. (VEGF promoter was used as the positive control).

HIF1α upregulates LASP-1 expression in xenograft mouse model

To determine whether HIF1α regulates LASP-1 expression in vivo, we injected Panc-1 cells subcutaneously into the right flank of nude nu/nu mice. When the tumors reached 100 mm3, the mice were intraperitoneally injected with saline or digoxin (2 mg/kg) on a daily basis to inhibit HIF activity (21, 24, 25). The tumor was palpable at 5 days after inoculation and all of the mice had developed tumors by the end of the experiment. Compared with the saline control group, the average tumor volume in the digoxin group was reduced obviously (P < 0.01; Fig. 4A–C). Next, we evaluated the association between HIF1α and LASP-1 by Western blot analysis (Fig. 4D) and immunohistochemistry (Fig. 4E) and the results suggest that expression of LASP-1 was decreased as a result of the HIF1α level reduction by digoxin.

Figure 4.

HIF1α promotes LASP-1 overexpression in a xenograft PDAC mouse model and in PDAC patient specimens. A, Panc-1 cells were subcutaneously implanted into the nude nu//nu mice, which were treated with saline or digoxin (2 mg/kg) every day. The data on all primary tumors are expressed as mean ± SD. B, representative images of mice injected with Panc-1 cells subcutaneously implanted into the nude mice (nu/nu). C, representative figure of tumors formed. D, HIF1α and LASP-1 expression determined by Western blot analysis in pancreatic cancer tissues of nude mice (nu/nu) treated by saline or digoxin. E, representative immunohistochemical staining of HIF1α and LASP-1 in pancreatic cancer tissues of nude nu/nu mice treated by saline or digoxin (magnification, ×200 and ×400). F, immunohistochemical analysis of HIF1α and LASP-1 correlative expression in human PDAC surgical samples (magnification, ×200). G, statistical analysis of immunohistochemical results of HIF1α and LASP-1 expression in human PDAC surgical samples. P values were analyzed by the χ2 test. H, the real distribution of immunohistochemical results between HIF1α and LASP-1 expression.

Figure 4.

HIF1α promotes LASP-1 overexpression in a xenograft PDAC mouse model and in PDAC patient specimens. A, Panc-1 cells were subcutaneously implanted into the nude nu//nu mice, which were treated with saline or digoxin (2 mg/kg) every day. The data on all primary tumors are expressed as mean ± SD. B, representative images of mice injected with Panc-1 cells subcutaneously implanted into the nude mice (nu/nu). C, representative figure of tumors formed. D, HIF1α and LASP-1 expression determined by Western blot analysis in pancreatic cancer tissues of nude mice (nu/nu) treated by saline or digoxin. E, representative immunohistochemical staining of HIF1α and LASP-1 in pancreatic cancer tissues of nude nu/nu mice treated by saline or digoxin (magnification, ×200 and ×400). F, immunohistochemical analysis of HIF1α and LASP-1 correlative expression in human PDAC surgical samples (magnification, ×200). G, statistical analysis of immunohistochemical results of HIF1α and LASP-1 expression in human PDAC surgical samples. P values were analyzed by the χ2 test. H, the real distribution of immunohistochemical results between HIF1α and LASP-1 expression.

Close modal

HIF1 correlates with LASP-1 expression in specimens of human PDAC

To determine whether HIF1 indeed regulates the expression of LASP-1 in patients with PDAC, we performed immunohistochemical staining to determine HIF1α and LASP-1 levels in PDAC specimens. As shown in Fig. 4F, LASP-1 expression colocalized with HIF1α in consecutive sections of PDAC tissues at different grades. Importantly, HIF1α expression in PDAC specimens significantly correlated with the levels of LASP-1 (Fig. 4G and H), implicating that HIF1α is a critical regulator for LASP-1 overexpression in patients with PDAC.

LASP-1 overexpression is able to rescue the inhibition of PDAC metastasis by HIF1 knockdown

To understand the role of LASP-1 in HIF1α-mediated migration, we ectopically expressed LASP-1 in HIF1α knockdown Panc-1 and CFPAC-1 cells. As shown in Fig. 5A (left, Panc-1; right, CFPAC-1), LASP-1 overexpression at least partially rescued the inhibitory effect of HIF1α knockdown on PDAC cell migration (P < 0.05), suggesting that LASP-1 was involved in HIF1α-mediated migration.

Figure 5.

LASP-1 is critical for PDAC metastasis in an orthotopic mouse model. A, Panc-1 (left) and CFPAC-1 (right) cells were cotransfected with siHIF1α (50 nmol/L) and pcDNA3.1-LASP-1 (2 μg) plasmids and assessed by Western blot (top) and Boyden chambers (bottom) analysis. The experiments were performed independently three times. *, P < 0.05 (magnification, ×200). B, the orthotopic xenograft pancreatic cancer mouse model using Panc-1/pLV Vector and Panc-1/pLV LASP-1 cells treated with saline or digoxin. Representative images of primary pancreatic tumor and metastatic tumors of liver, gut, and mesentery are shown. C, statistical analysis of the total number of visible metastatic lesions in liver, gut, and mesentery by the t test. Data are presented as mean ± SD. **, P < 0.01. D, hematoxylin and eosin staining verified the tumors from pancreas, liver, gut, and spleen (magnification, ×100).

Figure 5.

LASP-1 is critical for PDAC metastasis in an orthotopic mouse model. A, Panc-1 (left) and CFPAC-1 (right) cells were cotransfected with siHIF1α (50 nmol/L) and pcDNA3.1-LASP-1 (2 μg) plasmids and assessed by Western blot (top) and Boyden chambers (bottom) analysis. The experiments were performed independently three times. *, P < 0.05 (magnification, ×200). B, the orthotopic xenograft pancreatic cancer mouse model using Panc-1/pLV Vector and Panc-1/pLV LASP-1 cells treated with saline or digoxin. Representative images of primary pancreatic tumor and metastatic tumors of liver, gut, and mesentery are shown. C, statistical analysis of the total number of visible metastatic lesions in liver, gut, and mesentery by the t test. Data are presented as mean ± SD. **, P < 0.01. D, hematoxylin and eosin staining verified the tumors from pancreas, liver, gut, and spleen (magnification, ×100).

Close modal

To confirm the role for LASP-1 in pancreatic cancer cell invasion and metastasis in vivo, we developed an orthotopic pancreatic cancer mouse model using Panc-1/pLV Vector and Panc-1/pLV LASP-1 cells. When compared with Panc-1/pLV Vector cells, the morphology of Panc-1/pLV LASP-1 cells became irregular and had thin and long pseudopods (Supplementary Fig. S3A). The upregulation of LASP-1 protein in Panc-1/pLV LASP-1 cells was confirmed by Western blot analysis (Supplementary Fig. S3B). When injected orthotopically, Panc-1 cells developed primary tumor in the pancreas and distant metastases in the liver, gut, and mesentery over the course of 6 weeks. The size of primary pancreatic tumor and metastases of liver, gut, and mesentery in Panc-1/pLV Vector group was suppressed by digoxin treatment (mean ± SD: 9.17 ± 1.47 vs. 3.33 ± 1.03, P < 0.01; Fig. 5B and C). However, the Panc-1/pLV LASP-1 + digoxin group developed a significantly larger primary pancreatic tumor and higher number of metastatic lesions in liver, gut, and mesentery when compared with Panc-1/pLV Vector + digoxin tumors (mean ± SD: 3.33 ± 1.03 vs. 22.83 ± 2.79, P < 0.01; Fig. 5B and C). The tumors from pancreas, liver, gut, and spleen were further confirmed by hematoxylin and eosin staining (Fig. 5D). Taken together, these results suggested that overexpression of LASP-1 is critical for HIF1α-mediated PDAC metastasis.

LASP-1 correlated with lymph node metastasis in patients with PDAC

Our in vitro data suggest that LASP-1 overexpression may contribute to PDAC progression by promoting PDAC cell migration, invasion, and metastasis. To further critically examine this possibility, we evaluate the correlation between LASP-1 expression levels and clinicopathologic features and overall survival among our cohort of patients with PDAC (Table 1). There was no obvious correlation between expression of LASP-1 and age, gender, and histologic grade of patients with PDAC. However, LASP-1 expression was correlated with the pathologic tumor node metastasis stage (χ2 = 21.806, P < 0.05) and lymph node metastasis (χ2 = 17.481, P < 0.01) of PDAC samples (Table 1). Importantly, patients with PDAC with high or medium (+++ or ++) LASP-1 protein expression had significantly worse overall survival than those with negative or low (− or +) LASP-1 expression (P = 0.008; median time, 8 and 16 months; Fig. 6). Taken together, these data indicated that LASP-1 correlates with lymph node metastasis and influences the prognosis of patients with PDAC.

Figure 6.

LASP-1 influences the prognosis of patients with PDAC. Association between LASP-1 expression levels and the overall survival of patients with PDAC. Patients with PDAC (n = 91) were stratified into two groups according to LASP-1 immunohistochemical staining intensity. Patients with high LASP-1 expression (intensity grade ++ and +++) had much worse overall survival when compared to patients with low LASP-1 expression (intensity grade − and +). P = 0.008 was determined with the log-rank test.

Figure 6.

LASP-1 influences the prognosis of patients with PDAC. Association between LASP-1 expression levels and the overall survival of patients with PDAC. Patients with PDAC (n = 91) were stratified into two groups according to LASP-1 immunohistochemical staining intensity. Patients with high LASP-1 expression (intensity grade ++ and +++) had much worse overall survival when compared to patients with low LASP-1 expression (intensity grade − and +). P = 0.008 was determined with the log-rank test.

Close modal
Table 1.

Correlation of LASP-1 expression to clinicopathologic features in PDAC

LASP-1
Parameters++++++χ2P
Age, y     1.147 0.766 
 ≤60 10 12 23   
 >60 12 14   
Gender     1.913 0.591 
 Male 15 13 21   
 Female 16   
pTNM stage     21.806 0.010a 
 I   
 II 13 13 15   
 III 12   
 IV 10   
Histologic grade     5.023 0.541 
 G1 14   
 G2   
 G3 15   
LN metastasis     17.481 0.001a 
 N0 16 11   
 N1 10 30   
LASP-1
Parameters++++++χ2P
Age, y     1.147 0.766 
 ≤60 10 12 23   
 >60 12 14   
Gender     1.913 0.591 
 Male 15 13 21   
 Female 16   
pTNM stage     21.806 0.010a 
 I   
 II 13 13 15   
 III 12   
 IV 10   
Histologic grade     5.023 0.541 
 G1 14   
 G2   
 G3 15   
LN metastasis     17.481 0.001a 
 N0 16 11   
 N1 10 30   

NOTE: Statistical data of LASP-1 expression in relation to clinicopathologic features in PDAC surgical samples. Values of P were calculated by the χ2 test. N0 and N1 refer to the absence and presence of regional lymph node (LN) metastasis, respectively. pTNM stage refers to the pathologic tumor node metastasis (pTNM) stage.

aStatistically significant (P < 0.05).

In this study, we investigated the role of LASP-1 in PDAC progression and metastasis. Our data showed that LASP-1 expression levels were higher in PDAC than in adjacent nontumorous tissues. Intriguingly, LASP-1 overexpression was also observed in metastatic breast (13), ovarian (14), colorectal (15), and hepatocellular cancer tissues (26) and cell lines. Our mechanistic studies revealed that LASP-1 overexpression in PDAC was mainly mediated by HIF1α, which directly binds to a HRE on LASP-1 promoter. Importantly, HIF1α inhibition with digoxin drastically reduced LASP-1 protein levels in a PDAC xenograft mouse model. Moreover, immunohistochemical staining on consecutive sections of PDAC specimens indicated strong correlation between HIF1α levels and LASP-1 levels. These observations strongly support LASP-1 as a novel direct target gene of HIF1α in PDAC.

Hypoxia is commonly presented in the microenvironment of solid tumors (27). The constitutive expression of HIF1α in PDAC was also previously reported (28). Our data indicated that elevated HIF1α levels in PDAC transactivate LASP-1 gene transcription and protein expression, which in turn dysregulate the actin cytoskeleton in metastatic PDAC cells to promote invasion and metastasis. Intriguingly, HIF1α also transactivate the gene transcription of the prometastasis actin-bundling protein fascin-1 (23). Taken together, these findings indicated that HIF1α is a key regulator of the actin network remodeling in during cancer invasion and metastasis. By upregulating LASP-1 and fascin-1, HIF1α promotes the formation of membrane protrusions such as lamellipodia and filopodia, which provides driving forces for PDAC cell motility, invasiveness, and dissemination (29–32). In addition to the HIF1/LASP-1 signaling pathway, other pathways such as TGFβ (33), SLIT2-ROBO (34), and CXCL12/CXCR4 (35) signaling pathways, also influence PDAC metastasis. Understanding the interaction among these pathways may provide new clues for inhibiting metastasis of PDAC.

Our data indicated that LASP-1 might play a causal role in PDAC metastasis. Indeed, LASP-1 expression in patients with PDAC strongly associated with lymph node metastasis and poor clinical prognosis. Furthermore, we showed that LASP-1 indeed promoted the liver, gut, and mesentery metastases by using the orthotopic xenograft mouse model of pancreatic cancer. In summary, results from in vitro and in vivo experiments indicated that LASP-1 overexpression was a critical driver for PDAC cell migration and metastasis.

Thus, we demonstrated that LASP-1 was upregulated in PDAC particularly in those with metastasis, thereby indicating its relationship with poor clinical prognosis. LASP-1 stimulated cancer cell metastasis and aggressive phenotypes in vitro and in vivo. Most importantly, HIF1α regulated LASP-1 expression by binding to the HRE. Therefore, inhibiting LASP-1 expression may be more effective for treating metastatic PDAC.

No potential conflicts of interest were disclosed.

Conception and design: T. Zhao, H. Ren, S. Yang, J. Hao

Development of methodology: T. Zhao, H. Ren, J. Chen, Y. Sun, J. Sun, H. Zhang, J. Hao

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): T. Zhao, H. Ren, J. Li, J. Chen, W. Xin, Y. Sun, L. Sun, J. Sun, X. Wang, J. Hao

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): T. Zhao, H. Ren, J. Sun, C. Huang, H. Zhang, S. Yang, J. Hao

Writing, review, and/or revision of the manuscript: T. Zhao, H. Ren, J. Chen, J. Sun, S. Gao, S. Yang, J. Hao

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): T. Zhao, H. Ren, J. Chen, H. Zhang, Y. Sun, S. Gao, J. Hao

Study supervision: H. Ren, Y. Sun, J. Hao

Other (performed the immunohistochemistry experiments and prepared the regents and buffer): Y. Yang

This work was supported by the National Natural Science Foundation of China (grants 81302082, 81272685, 31301151, 81172355, 31471340, 31470957, 81472264, and 81401957), the Key Program of Natural Science Foundation of Tianjin (grants11JCZDJC18400 and 13YCYBYC37400), and the Major Anticancer Technologies R&D Program of Tianjin (grant 12ZCDZSY16700). S. Yang is supported by the NIH (grant R01CA175741).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1.
Tan
CR
,
Yaffee
PM
,
Jamil
LH
,
Lo
SK
,
Nissen
N
,
Pandol
SJ
, et al
Pancreatic cancer cachexia: a review of mechanisms and therapeutics
.
Front Physiol
2014
;
5
:
88
.
2.
Siegel
R
,
Ma
J
,
Zou
Z
,
Jemal
A
. 
Cancer statistics, 2014
.
CA Cancer J Clin
2014
;
64
:
9
29
.
3.
Vincent
A
,
Herman
J
,
Schulick
R
,
Hruban
RH
,
Goggins
M
. 
Pancreatic cancer
.
Lancet
2011
;
378
:
607
20
.
4.
Spivak-Kroizman
TR
,
Hostetter
G
,
Posner
R
,
Aziz
M
,
Hu
C
,
Demeure
MJ
, et al
Hypoxia triggers hedgehog-mediated tumor-stromal interactions in pancreatic cancer
.
Cancer Res
2013
;
73
:
3235
47
.
5.
Chew
CS
,
Chen
X
,
Parente
JA
 Jr.
,
Tarrer
S
,
Okamoto
C
,
Qin
HY
. 
Lasp-1 binds to non-muscle F-actin in vitro and is localized within multiple sites of dynamic actin assembly in vivo
.
J Cell Sci
2002
;
115
:
4787
99
.
6.
Tomasetto
C
,
Moog-Lutz
C
,
Regnier
CH
,
Schreiber
V
,
Basset
P
,
Rio
MC
. 
Lasp-1 (MLN 50) defines a new LIM protein subfamily characterized by the association of LIM and SH3 domains
.
FEBS Lett
1995
;
373
:
245
9
.
7.
Tomasetto
C
,
Regnier
C
,
Moog-Lutz
C
,
Mattei
MG
,
Chenard
MP
,
Lidereau
R
, et al
Identification of four novel human genes amplified and overexpressed in breast carcinoma and localized to the q11-q21.3 region of chromosome 17
.
Genomics
1995
;
28
:
367
76
.
8.
Schreiber
V
,
Moog-Lutz
C
,
Regnier
CH
,
Chenard
MP
,
Boeuf
H
,
Vonesch
JL
, et al
Lasp-1, a novel type of actin-binding protein accumulating in cell membrane extensions
.
Mol Med
1998
;
4
:
675
87
.
9.
Keicher
C
,
Gambaryan
S
,
Schulze
E
,
Marcus
K
,
Meyer
HE
,
Butt
E
. 
Phosphorylation of mouse LASP-1 on threonine 156 by cAMP- and cGMP-dependent protein kinase
.
Biochem Biophys Res Commun
2004
;
324
:
308
16
.
10.
Traenka
C
,
Remke
M
,
Korshunov
A
,
Bender
S
,
Hielscher
T
,
Northcott
PA
, et al
Role of LIM and SH3 protein 1 (LASP1) in the metastatic dissemination of medulloblastoma
.
Cancer Res
2010
;
70
:
8003
14
.
11.
Rachlin
AS
,
Otey
CA
. 
Identification of palladin isoforms and characterization of an isoform-specific interaction between Lasp-1 and palladin
.
J Cell Sci
2006
;
119
:
995
1004
.
12.
Grunewald
TG
,
Butt
E
. 
The LIM and SH3 domain protein family: structural proteins or signal transducers or both?
Mol Cancer
2008
;
7
:
31
.
13.
Grunewald
TG
,
Kammerer
U
,
Schulze
E
,
Schindler
D
,
Honig
A
,
Zimmer
M
, et al
Silencing of LASP-1 influences zyxin localization, inhibits proliferation and reduces migration in breast cancer cells
.
Exp Cell Res
2006
;
312
:
974
82
.
14.
Grunewald
TG
,
Kammerer
U
,
Winkler
C
,
Schindler
D
,
Sickmann
A
,
Honig
A
, et al
Overexpression of LASP-1 mediates migration and proliferation of human ovarian cancer cells and influences zyxin localisation
.
Br J Cancer
2007
;
96
:
296
305
.
15.
Zhao
L
,
Wang
H
,
Liu
C
,
Liu
Y
,
Wang
X
,
Wang
S
, et al
Promotion of colorectal cancer growth and metastasis by the LIM and SH3 domain protein 1
.
Gut
2010
;
59
:
1226
35
.
16.
Wang
B
,
Feng
P
,
Xiao
Z
,
Ren
EC
. 
LIM and SH3 protein 1 (Lasp1) is a novel p53 transcriptional target involved in hepatocellular carcinoma
.
J Hepatol
2009
;
50
:
528
37
.
17.
Han
ZB
,
Ren
H
,
Zhao
H
,
Chi
Y
,
Chen
K
,
Zhou
B
, et al
Hypoxia-inducible factor (HIF)-1 alpha directly enhances the transcriptional activity of stem cell factor (SCF) in response to hypoxia and epidermal growth factor (EGF)
.
Carcinogenesis
2008
;
29
:
1853
61
.
18.
Zhao
T
,
Gao
S
,
Wang
X
,
Liu
J
,
Duan
Y
,
Yuan
Z
, et al
Hypoxia-inducible factor-1alpha regulates chemotactic migration of pancreatic ductal adenocarcinoma cells through directly transactivating the CX3CR1 gene
.
PLoS ONE
2012
;
7
:
e43399
.
19.
Yang
S
,
Zhang
JJ
,
Huang
XY
. 
Mouse models for tumor metastasis
.
Methods Mol Biol
2012
;
928
:
221
8
.
20.
Yang
S
,
Huang
XY
. 
Ca2+ influx through L-type Ca2+ channels controls the trailing tail contraction in growth factor-induced fibroblast cell migration
.
J Biol Chem
2005
;
280
:
27130
7
.
21.
Chaturvedi
P
,
Gilkes
DM
,
Wong
CC
,
Kshitiz
,
Luo
W
,
Zhang
H
, et al
Hypoxia-inducible factor-dependent breast cancer-mesenchymal stem cell bidirectional signaling promotes metastasis
.
J Clin Invest
2013
;
123
:
189
205
.
22.
Fujisawa
T
,
Joshi
B
,
Nakajima
A
,
Puri
RK
. 
A novel role of interleukin-13 receptor alpha2 in pancreatic cancer invasion and metastasis
.
Cancer Res
2009
;
69
:
8678
85
.
23.
Zhao
X
,
Gao
S
,
Ren
H
,
Sun
W
,
Zhang
H
,
Sun
J
, et al
Hypoxia-inducible factor-1 promotes pancreatic ductal adenocarcinoma invasion and metastasis by activating transcription of the actin-bundling protein fascin
.
Cancer Res
2014
;
74
:
2455
64
.
24.
Zhang
H
,
Wong
CC
,
Wei
H
,
Gilkes
DM
,
Korangath
P
,
Chaturvedi
P
, et al
HIF-1-dependent expression of angiopoietin-like 4 and L1CAM mediates vascular metastasis of hypoxic breast cancer cells to the lungs
.
Oncogene
2012
;
31
:
1757
70
.
25.
Wong
CC
,
Zhang
H
,
Gilkes
DM
,
Chen
J
,
Wei
H
,
Chaturvedi
P
, et al
Inhibitors of hypoxia-inducible factor 1 block breast cancer metastatic niche formation and lung metastasis
.
J Mol Med
2012
;
90
:
803
15
.
26.
Wang
H
,
Li
W
,
Jin
X
,
Cui
S
,
Zhao
L
. 
LIM and SH3 protein 1, a promoter of cell proliferation and migration, is a novel independent prognostic indicator in hepatocellular carcinoma
.
Eur J Cancer
2013
;
49
:
974
83
.
27.
Yang
Y
,
Sun
M
,
Wang
L
,
Jiao
B
. 
HIFs, angiogenesis, and cancer
.
J Cell Biochem
2013
;
114
:
967
74
.
28.
Akakura
N
,
Kobayashi
M
,
Horiuchi
I
,
Suzuki
A
,
Wang
J
,
Chen
J
, et al
Constitutive expression of hypoxia-inducible factor-1alpha renders pancreatic cancer cells resistant to apoptosis induced by hypoxia and nutrient deprivation
.
Cancer Res
2001
;
61
:
6548
54
.
29.
Sun
J
,
He
H
,
Pillai
S
,
Xiong
Y
,
Challa
S
,
Xu
L
, et al
GATA3 transcription factor abrogates Smad4 transcription factor-mediated fascin overexpression, invadopodium formation, and breast cancer cell invasion
.
J Biol Chem
2013
;
288
:
36971
82
.
30.
Sun
J
,
He
H
,
Xiong
Y
,
Lu
S
,
Shen
J
,
Cheng
A
, et al
Fascin protein is critical for transforming growth factor beta protein-induced invasion and filopodia formation in spindle-shaped tumor cells
.
J Biol Chem
2011
;
286
:
38865
75
.
31.
Chen
L
,
Yang
S
,
Jakoncic
J
,
Zhang
JJ
,
Huang
XY
. 
Migrastatin analogues target fascin to block tumour metastasis
.
Nature
2010
;
464
:
1062
6
.
32.
Yang
S
,
Huang
FK
,
Huang
J
,
Chen
S
,
Jakoncic
J
,
Leo-Macias
A
, et al
Molecular mechanism of fascin function in filopodial formation
.
J Biol Chem
2013
;
288
:
274
84
.
33.
Ostapoff
KT
,
Cenik
BK
,
Wang
M
,
Ye
R
,
Xu
X
,
Nugent
D
, et al
Neutralizing murine TGFbetaR2 promotes a differentiated tumor cell phenotype and inhibits pancreatic cancer metastasis
.
Cancer Res
2014
;
74
:
4996
5007
.
34.
Gohrig
A
,
Detjen
KM
,
Hilfenhaus
G
,
Korner
JL
,
Welzel
M
,
Arsenic
R
, et al
Axon guidance factor SLIT2 inhibits neural invasion and metastasis in pancreatic cancer
.
Cancer Res
2014
;
74
:
1529
40
.
35.
Singh
AP
,
Arora
S
,
Bhardwaj
A
,
Srivastava
SK
,
Kadakia
MP
,
Wang
B
, et al
CXCL12/CXCR4 protein signaling axis induces sonic hedgehog expression in pancreatic cancer cells via extracellular regulated kinase- and Akt kinase-mediated activation of nuclear factor kappaB: implications for bidirectional tumor-stromal interactions
.
J Biol Chem
2012
;
287
:
39115
24
.