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 breast cancer patients,

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 HIF-1α, which directly binds to and

transactivats the LASP-1 promoter. Our findings indicate that LASP-1 is a novel direct

HIF-1α target gene that promotes PDAC metastasis and progression.

Materials and methods

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 Chinese Academy of Sciences (Shanghai, China) and MIA-

PaCa-2 was obtained from the American Type Culture Collection. 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 Dulbecco’s modified Eagle medium (DMEM) with 10% fetal bovine

serum (FBS). For hypoxic treatment, cells were placed in a modulator incubator (Thermo

Electron Co., Forma, MA, USA) in an atmosphere consisting of 93.5% N2, 5%CO2, and

1.5% O

2.

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 HIF-1α (1:1000),

LASP-1 (1:2000), and β-actin (1:1000) (Table S1). Specific proteins were visualized using

an enhanced chemiluminescence detection reagent (Pierce).

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 RT-PCR

(Takara). Each sample was processed in triplicate, and β-actin was used as loading control.

Each experiment was repeated independently for at least three times. PCR primers used are

indicated in 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 min, permeabilized with 0.1% Triton X-100 in PBS for 30

min at room temperature, and blocked for 1 h 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 (Beyotine 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 (HREs) of the VEGF promoter were used as a positive control (17, 18).

The PCR primers are indicated in Table S1.

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

Small interfering RNAs (siRNAs) against LASP-1 and HIF-1α were designed and

synthesized from GenePharma (Shanghai, China) (Table S1). The human LASP-1 cDNA

was cloned into the pcDNA3.1 plasmid expression vector. The pcDNA3.1-HIF-1α 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). Following transfection, the

medium containing Lentivirus was collected, filtered, and transferred onto Panc-1 cells.

Infected cells were selected with puromycin (1μg/mL) for 7 days.

Genomic DNA fragments of the human LASP1 gene, spanning from +1 to ?2000 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 h. The cells were collected for cell migration

and invasion analysis, western blot analysis, and RT-PCR, immunofluorescence, etc.

A wound healing assay was performed according to published protocol (20). Invasion assays

were performed with 8.0μm pore inserts in a 24-well Trans-well. For this assay, 1×105 cells

were isolated and added to the upper chamber of a trans-well with DMEM. The invasion

assay was performed using 1/6 diluted matrigel (BD Bioscience)-coated filters. DMEM with

10% fetal bovine serum was added to the lower 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 for 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 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. Cells were harvested by trypsinization, washed in PBS,

resuspended at 107 cells/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 3 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). 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 years to 83 years) undergoing surgical resection with histologic diagnosis of PDAC

at the Tianjin Cancer Institute and Hospital. Immunohistochemistry for HIF-1α and LASP-1

of PDAC patient tissues was performed using a DAB substrate kit (Maxin, Fuzhou, China).

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 (+++).

Student’s t-test for paired data was used to compare mean values. ANOVA is used to

analysis two groups’ data with continuous variables. Non-parametric data were analyzed

with Mann-Whitney U test. The categorical data was analyzed by either Fisher’s exact or

Chi-Square method. Each experiment was conducted independently for at least three times,

and values were presented as mean ± standard deviation (SD), unless otherwise stated.

Analyses were performed using SPSS18.0 statistical analysis software.

Results

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 inter-individual variations, LASP-1 protein (Figure 1A) and mRNA (Figure 1B)

levels were found to be evidently upregulated in PDAC samples when compared to 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 (Figure 1C).

Intriguingly, in pancreas tumors other than malignant PDAC, such as benign tumors: serous

cystadenoma and neuroendocrine tumor, LASP-1 expression was negative (Figure 1C).

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 knockdown LASP-1 expression in two PDAC cell

lines with high endogenous LASP-1 levels (CFPAC-1 and MIA-PaCa-2) (Figure 2A). Out

of the three pairs of siRNA, siRNA #3 most efficiently knockdown LASP-1 expression by

more than 70% (Figure 2B, 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 (Figure 2D).

In the wound-healing assays, the migratory activity of CFPAC-1 and MIA-PaCa-2 cells was

also inhibited by LASP-1 silencing (Figure 2E). Immunofluorescence analysis confirmed

regional co-localization between LASP-1 and F-actin (Figure 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 (Figure 2F). These data

indicated that LASP-1 was critical for the migration and invasion of PDAC cells.

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) (Figure 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 (Figure 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 (Figure 2H). Importantly, BxPC-3/pcDNA3.1-LASP1

and Panc-1/pcDNA3.1-LASP1 displayed some pseudopods extending from cell bodies

promote PDAC cell migration and invasion.

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

Our previous study indicated HIF-1 as a critical transcriptional factor in pancreatic cancer

cell migration (18, 23). To determine whether HIF-1 regulates transcription of LASP-1 in

pancreatic cancer cells, we used specific siRNAs targeting HIF-1α to effectively reduce

HIF-1α expression (Figure S2). Knockdown of HIF-1α expression decreased LASP-1

mRNA (Figure 3A, left) and protein (Figure 3A, right) expression (p<0.05). Moreover,

when HIF-1α was overexpressed in Panc-1 cells, LASP-1 mRNA (Figure 3B, left) and

protein (Figure 3B, right) expression markedly increased (p<0.05). Taken together, these

results suggest that HIF-1α plays a critical role in LASP-1 expression.

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 Figure 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

survey the promoter region of human LASP-1 gene and identified 4 hypoxia response

elements (HREs) (Figure 3D).

To investigate whether HIF-1α 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-HIF-1α antibody, only the HRE of LASP-1

promoter located at ?1005 to ?1001 was detected (Figure 3E, upper). The fragment

immunoprecipitated by anti-HIF-1α antibody significantly increased (Figure 3E, lower,

p<0.01) under hypoxia, suggesting that hypoxia promoted the binding of HIF-α to LASP-1

promoter.

To determine whether the binding of HIF-1α activates LASP-1 promoter, we constructed a

full-length LASP-1 luciferase promoter vector (containing HREs, ?1005 to ?1001) and co-

transfected this reporter construct with or without HIF-1α cDNA into Panc-1 cells.

Luciferase analysis showed that HIF-1α overexpression (pcDNA-HIF1α) significantly

increased LASP-1 promoter activity in Panc-1 cells (~2.9-fold, p<0.05) (Figure 3F). To

determine whether the HRE1 site is required for HIF-1α to transactivate LASP-1 promoter,

this HIF-1 α binding site was mutated from ACGTG to ACATG. As shown in Figure 3F,

the mutation of HRE1 almost abolished the transactivation of LASP-1 promoter by HIF-1α.

(VEGF promoter was used as the positive control).

HIF-1α upregulates LASP-1 expression in xenograft mouse model

To determine whether HIF-1α 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

HIF activity (21, 24, 25). The tumor was palpable at 5 days after inoculation and that all of

the mice had developed tumors by the end of the experiment. Compared with saline control

group, the average tumor volume in digoxin group was reduced obviously (p<0.01) (Figure

4A, B and C). Next, we evaluated the association between HIF-1α and LASP-1 by Western

blot (Figure 4D) and immunohistochemistry (Figure 4E) and the results suggest that

expression of LASP-1 was decreased as a result of the HIF-1α level reduction by digoxin. HIF-1 correlates with LASP-1 expression in the specimens of human PDAC

To determine whether HIF-1 indeed regulates the expression of LASP-1 in PDAC patients,

we performed immunohistochemical staining to determine HIF-1α and LASP-1 levels in

PDAC specimens. As shown in Figure 4F, LASP-1 expression co-localizated with HIF-1α

in consecutive sections of PDAC tissues at different grades. Importantly, HIF-1α expression

in PDAC specimens significantly correlated with the levels of LASP-1 (Figure 4G and 4H),

implicating that HIF-1α is a critical regulator for LASP-1 overexpression in PDAC patients. LASP-1 overexpression is able to rescue the inhibition of PDAC metastasis by HIF-1 knockdown

To understand the role of LASP-1 in HIF-1α-mediated migration, we ectopically expressed

LASP-1 in HIF-1α knockdown Panc-1 and CFPAC-1 cells. As shown in Figures 5A

(Panc-1: left; CFPAC-1: right), LASP-1 overexpression at least partially rescued the

inhibitory effect of HIF-1α knockdown on PDAC cell migration (p<0.05), suggesting that

LASP-1 was involved in HIF-1α-mediated migration.

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 have the thin and long pseudopods

(Figure S3A). The up-regulation of LASP-1 protein in Panc-1/pLV LASP-1 cells was

confirmed by western blot experiment (Figure 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) versus (3.33±1.03, p<0.01) (Figure 5B, 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) versus (22.83±2.79,

p<0.01) (Figure 5B, C). The tumors from pancreas, liver, gut and spleen were further

confirmed by hematoxylin-eosin staining (Figure 5D). Taken together, these results

suggested that overexpression of LASP-1 is critical for HIF-1α mediated PDAC metastasis. LASP-1 correlated with lymph node metastasis in PDAC patients

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

1). There was no obvious correlation between expression of LASP-1 and age, gender and

histological grade of PDAC patients. However, LASP-1 expression was correlated with the

pathological 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, PDAC patients 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) (Figure 6). Taken together, these data indicated that LASP-1 correlates with lymph

node metastasis and influences the prognosis of PDAC patients.

Discussion

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 non-

tumorous tissues. Intriguingly, LASP-1 overexpression was also observed in metastatic

breast (13), ovarian (14), colorectal (15), and hepatocellular cancer (26) tissues and cell

lines. Our mechanistic studies revealed that LASP-1 overexpression in PDAC was mainly

mediated by HIF-1α, which directly binds to a hypoxia response element on LASP-1

promoter. Importantly, HIF-1α inhibition with digoxin drastically reduced LASP-1 protein

levels in a PDAC xenograft mouse model. Moreover, IHC staining on consecutive sections

of PDAC specimens indicated strong correlation between HIF-1α levels and LASP-1 levels.

These observations strongly support LASP-1 as a novel direct target gene of HIF-1α in

PDAC.

Hypoxia is commonly presented in the microenvironment of solid tumors (27). The

constitutive expression of HIF-1α in PDAC was also previously reported (28). Our data

indicated that elevated HIF-1α levels in PDAC transactivates LASP-1 gene transcription and

protein expression, which in turn dysregulate the actin cytoskeleton in metastatic PDAC

cells to promote invasion and metastasis. Intriguingly, HIF-1α also transactivate the gene

transcription of the pro-metastasis actin-bundling protein fascin-1 (23). Taken together,

these findings indicated that HIF-1α is a key regulator of the actin network remodeling in

during cancer invasion and metastasis. By up-regulating LASP-1 and fascin-1, HIF-1α

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 HIF-1/LASP-1 signaling pathway, other pathways such as TGF-β (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 PDAC patients 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.

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, HIF-1α regulated LASP-1 expression by binding to the HRE. Therefore,

inhibiting LASP-1 expression may be more effective for treating metastatic PDAC. Supplementary Material

Refer to Web version on PubMed Central for supplementary material.

Acknowledgments

National Natural Science Foundation of China (Grant No. 81302082, 81272685, 31301151, 81172355, 31471340,

31470957, 81472264, 81401957); Key Program of Natural Science Foundation of Tianjin (Grant No.

11JCZDJC18400,13YCYBYC37400); Major Anticancer Technologies R & D Program of Tianjin (Grant No.

12ZCDZSY16700). S. Yang is supported by NIH (R01CA175741).

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

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 experiment. (B, C) LASP-1 protein expression in CFPAC-1 and MIA-PaCa2 cells transfected with negative control siRNA (siNC) and LASP-1 siRNA (siLASP-1#1-3) (50 nM) for 48 hours was determined by Western blot. β-actin was used as a loading control.

(D) Comparison of migration and invasion potential of CFPAC-1 (upper) and MIA-PaCa2 (lower) cells transfected with negative control (NC) siRNA and LASP-1 siRNA#3 (50 nM) for 48 hours using Boyden chambers. The experiments were performed independently for 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 NC siRNA and LASP-1 siRNA#3 (50 nM) 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 nM) stained for LASP-1 (red), F-actin (green), and 4′,6-diamidino-2-phenylindole (DAPI; blue) (magnification, 600×). Co-localization was indicated by the merged images showing yellow immunofluorescence. (G) Comparison of the migration and invasion potential of BxPC-3 (upper) and Panc-1 (lower) cells transfected with pcDNA3.1

localization was indicated by the merged images showing yellow immunofluorescence.

Figure 3. LASP-1 is a novel HIF-1α target gene critical for HIF-1α induced cell migration (A) CFPAC-1 cells were transfected with siHIF-1α #3 (50 nM) for 48 h and assessed HIF-1α and LASP-1 expression by qRT-PCR (left) and Western blot (right) analysis. The experiments were performed independently for three times. *p<0.05 versus control. (B) Panc-1 cells were transfected with pcDNA3.1-HIF1α plasmids (2 μg) for 48 hours and

assessed HIF-1α and LASP-1 expression by qRT-PCR (left) and Western blot (right) analysis. The experiments were performed independently for three times. *p<0.05 versus control. (C) HIF-1α and LASP-1 expression determined by Western blot analysis on four human PDAC cells cultured under normoxia (21% O 2) and hypoxia (1.5% O 2) for 12 hours. (N: Normoxia; H: Hypoxia). (D) The DNA sequence of the LASP-1 promoter. Four HRE sites are located at the different site. (E) Upper: Chromatin immunoprecipitation analysis HIF-1 binding to LASP-1 promoter in Panc-1 cells. 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. Lower: The comparison of gray value between normoxia and hypoxia in HRE1. **p <0.01. The experiments were performed independently for three times. (F) Luciferase analysis in Panc-1 cells. Panc-1 cells overexpressing HIF-1α

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 for three times.

Figure 4. HIF-1α promotes LASP-1 overexpression in xenograft PDAC mouse model and PDAC

patients specimens

(A) Panc-1 cells were subcutaneously implanted into the nude nu//nu mice, which were treated with saline or digoxin (2 mg/kg) everyday. The data of 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) HIF-1α 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 HIF-1α and LASP-1 in pancreatic cancer tissues of nude nu//nu mice treated by saline or digoxin. (magnification, 200× and 400×). (F)

immunohistochemical results between HIF-1α and LASP-1 expression.

Figure 5. LASP-1 is critical for PDAC metastasis in a orthotopic mouse model (A) Panc-1(left) and CFPAC-1(right) cells were co-transfected with siHIF1α(50 nM) and pcDNA3.1-LASP-1 (2 μg) plasmids and assessed by Western blot (upper) and Boyden

chambers. (lower) analysis. The experiments were performed independently for 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 were treated with saline or digoxin. Representative images of primary pancreatic tumor and metastatic tumors of liver, gut and mesentery were shown. (C) Statistical analysis of the total number of visible metastatic lesions in liver, gut and mesentery by t-test. Data were presented as mean±SD. **p <0.01. (D) Hematoxylin-eosin staining verified the tumors from pancreas, liver, gut and spleen. (magnification, 100×).

Figure 6. LASP-1 influences the prognosis of PDAC patients Association between LASP-1 expression levels and the overall survival of PDAC patients.

PDAC patients (n=91) were stratified into two groups according to LASP-1 IHC 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.

NIH-PA Author Manuscript

NIH-PA Author Manuscript NIH-PA Author Manuscript T

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