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. Author manuscript; available in PMC: 2017 Oct 1.
Published in final edited form as: Clin Cancer Res. 2016 Apr 28;22(19):4934–4946. doi: 10.1158/1078-0432.CCR-15-2780

Transforming Growth Factor-β Limits Secretion of Lumican by Activated Stellate Cells within Primary Pancreatic Adenocarcinoma Tumors

Ya’an Kang 1, David Roife 2, Yeonju Lee 3, Hailong Lv 4, Rei Suzuki 5, Jianhua Ling 6, Mayrim V Rios Perez 1, Xinqun Li 1, BingBing Dai 1, Michael Pratt 1, Mark J Truty 7, Deyali Chatterjee 8, Huamin Wang 9, Ryan M Thomas 10, Yu Wang 11, Eugene J Koay 3, Paul J Chiao 4, Matthew H Katz 1, Jason B Fleming 1
PMCID: PMC5127652  NIHMSID: NIHMS830323  PMID: 27126993

Abstract

Purpose

Pancreatic ductal adenocarcinoma (PDAC) is lethal cancer whose primary tumor is characterized by dense composition of cancer cells, stromal cells and extracellular matrix (ECM) composed largely of collagen. Within the PDAC tumor microenvironment, activated pancreatic stellate cells (PSC) are the dominant stromal cell type and responsible for collagen deposition. Lumican is a secreted proteoglycan that regulates collagen fibril assembly. We have previously identified that the presence of lumican in the ECM surrounding PDAC cells is associated with improved patient outcome after multimodal therapy and surgical removal of localized PDAC.

Experimental Design

Lumican expression in PDAC from 27 patients was determined by IHC and quantitatively analyzed for co-localization with PSC. In vitro studies examined the molecular mechanisms of lumican transcription and secretion from PSC (HPSC, HPaSteC), and cell adhesion and migration assays examined the effect of lumican on PSC in a collagen-rich environment.

Results

Here we identify PSC as a significant source of extracellular lumican production through quantitative IHC analysis. We demonstrate that the cytokine, transforming growth factor-β (TGF-β), negatively regulates lumican gene transcription within HPSC through its canonical signaling pathway and binding of SMAD4 to novel SBEs identified within the promoter region. Additionally, we found that the ability of HPSC to produce and secrete extracellular lumican significantly enhances HPSC adhesion and mobility on collagen.

Conclusion

Our results demonstrate that activated pancreatic stellate cells within PDAC secrete lumican under the negative control of TGF-β; once secreted, the extracellular lumican enhances stellate cell adhesion and mobility in a collagen rich environment.

Keywords: Lumican, Pancreatic Stellate Cells, TGF-β, Extra Cellular Matrix, Adhesion, Migration

Introduction

Pancreatic ductal adenocarcinoma (PDAC) remains a leading cause of cancer death and an unsolved health care dilemma in the United States (1, 2). Histologically, cancer cells comprise only a fraction of the PDAC tumor mass with the majority consisting of a desmoplastic fibrotic network of activated fibroblasts and pancreatic stellate cells (PSC), leukocytes, and ECM components (3–6). It is well recognized that the microenvironment plays an important role in pancreatic cancer cell survival, metastatic dissemination, and resistance to therapy (3, 7), but the mechanisms of this influence are poorly understood.

Lumican, a member of the small leucine-rich proteoglycan family (SLRP), is present within primary and metastatic tumors derived from various human malignancies, including pancreatic cancer (8–10). It is a secreted, collagen-binding ECM protein that is highly expressed in connective tissue throughout the body. The protein moiety of lumican binds collagen fibrils and the hydrophilic glycosaminoglycans regulate interfibrillar spacing (11–14) and collagen assembly (15, 16). Its complex and diverse proteoglycan structure suggests that lumican influences cell function through a variety of mechanisms (17, 18). We have previously demonstrated that the presence of lumican in the ECM surrounding, but not within, PDAC cells is associated with improved patient outcome after multimodal therapy and surgical removal of localized PDAC (19). The cell source and the molecular mechanisms controlling the secretion of lumican into the ECM of PDAC and other solid tumors are currently unknown.

Stellate cells are found in solid organs throughout the body [16], and pancreatic stellate cells were first isolated in 1998 by Apte et al [17]. Pancreatic stellate cells comprise 47% of pancreatic parenchyma and normally maintain tissue architecture in health and injury states through ECM protein synthesis and degradation. Pancreatic stellate cells usually exist in a quiescent state, [18], but become activated in response to injury. Activation of pancreatic stellate cells is marked by alpha-smooth muscle actin (α-SMA) expression and secretion of fibrillar collagen as part of an intense fibrosis until healing occurs when the stellate cells return to the quiescent state. Within the PDAC tumor microenvironment, activated stellate cells (PSC) are the dominant stromal cell type, and PSC have been demonstrated as the cell source of fibrillar collagen within the desmoplastic ECM of PDAC tumors (20). Recent studies focusing on tumor-stroma interaction suggest that stroma in PDAC acts to restrain cancer cell growth (21) with the depletion of α-SMA positive myofibroblasts (PSC) in the pancreas of genetically engineered mice resulting in aggressive cancer formation and reduced survival (22, 23).

The pleiotropic cytokine, transforming growth factor-β (TGF-β) is frequently present in the tumor microenvironment; it is one of the strongest inducers of ECM production during fibrogenesis (24, 25) and desmoplasia within pancreatic carcinoma (26). When signaling via its canonical pathway, TGF-β binds to TGF-β type II receptors, which phosphorylate TGF-β type I receptors and in turn phosphorylate cytoplasmic SMAD2 and SMAD3. The phosphorylated SMAD protein complex then binds to SMAD4, and is translocated into the nucleus. Once inside the nucleus, the complex binds to sites within gene promoter regions termed SMAD-binding elements (SBEs) in order to regulate transcription. Specific DNA sites identified as SBEs include CAGACA and the 8-bp palindromic sequence, GTCTAGAC (27–29). Both cancer and host cells within the tumor microenvironment are sources of TGF-β (30), but the specific interactions between TGF-β and different host cell types are incompletely understood.

Although suggested by previous observations (31), the molecular mechanisms controlling lumican secretion in human pancreatic cancer have not been described. In this study, we demonstrate that extracellular lumican is physically co-localized with PSC within the collagen dense ECM of primary PDAC tumors. When cultured, these PSC secrete large amounts of lumican and this production is controlled at the transcription level by the TGF-β-SMAD4 signaling axis. This is accomplished through SMAD4 binding to novel SBEs we have identified within the promoter region of the lumican gene. Once secreted, lumican in the extracellular space facilitates the adhesion and migration of PSC within a collagen rich environment.

Material and methods

Ethics statement

The xenograft protocol was approved by The University of Texas MD Anderson Cancer Center Institutional Review Board under #LAB07-0854. Excess patient tumor was collected only after the planned surgical resection and pathologic examination were complete. Patient specimens from pancreatectomies performed between 2009 and 2011 at The University of Texas MD Anderson Cancer Center were selected after reviewing medical records and tissue specimens.

Cell lines and reagents

Immortalized human pancreatic stellate cells (HPSC) harvested from primary PDAC tumors were a gift from Dr. Rosa F. Hwang (32), Immortalized normal human pancreatic ductal epithelial (HPNE) and 293T cells were a gift from Dr. Paul J. Chiao (UTMDACC). Human Pancreatic Fibroblasts (HPF) were purchased from Vitro Biopharma (#SC00A5). PANC-1, BxPC-3, and MiaPaCa-2 cell lines were obtained from ATCC. MDA-PATC43, 50, 53, and 66 cell lines were derived in our laboratory (33, 34). These cells lines were verified as human and unique by DNA fingerprinting, and all cell lines are authenticated by short tandem repeat (STR) DNA profiling at the Characterized Cell Line Core Facility of the MD Anderson Cancer Center within 6 months. All cell lines were maintained in Dulbecco’s modified Eagle’s medium (Life Technologies) supplemented with 10% FBS at 37°C in a 5% CO2. Human recombinant TGF-β and lumican (rLUM) were purchased from R&D Systems (#240-B and 2846-LU).

Immunohistochemistry (IHC), immunofluorescence staining (IF), and Vectra multispectral analysis

A tissue microarrays (TMA) was constructed using core samples from paraffin-embedded blocks of 27 PDAC primary tumors surgically removed at MD Anderson. These were prepared for IHC study at the Clinical Core Laboratory of MD Anderson. All reactions were visualized with 3,3′-diaminobenzidine (DAB) as a chromogen. Isotype controls for all antibodies were negative. The TMA slides with IHC stains were scanned using the Vectra slide scanner (PerkinElmer) to identify lumican expression signal (DAB stains), a representative digitized image was imported to Nuance (PerkinElmer) and the spectral library of hematoxylin and DAB was created. Then, InForm image analysis software (PerkinElmer) was applied to quantify LUM expression in stroma or tumor. The software was then employed to quantify DAB intensity in specific tissue classifiers (35, 36).

For double color IF, HPSC were seeded into 8-well Lab-Tek II chamber slides (#154534, Thermo Fisher Scientific), fixed and permeabilized for 10 minutes next day. The chamber slides were blocked with 1% BSA for 30 minutes, and the mixture of anti-lumican and anti-SMAD4 antibodies (5 ng/μl) were added and incubated overnight at 4°C. TMA slides were deparaffinized and rehydrated, then the paired mixtures of lumican with α-SMA primary antibodies (5 ng/μl) were incubated overnight at 4°C. Secondary antibodies either conjugated with Alexa Fluor 488 or with Alexa Fluor 594 of different species, depending on primary antibodies, which were purchased from Life Technologies (#A11008, A11012, and A11005). 2 ng/μl of these antibodies were incubated with the slides for 30 minutes at room temperature, and the slides were counterstained with DAPI (#D8417, Sigma-Aldrich) for 10 minutes, and mounted with DAKO Fluorescent mounting medium (#S3023, Dako). As a negative control, HPSC cells or TMA slides were applied with IgG.

Images were captured with an Olympus FV1000 Laser Scanning Confocal microscope and analyzed with FlowView software at the Flow Cytometry and Cellular Imaging Facility of MD Anderson. All images were captured with the same exposure time on all samples. These profiles were then used to discriminate the individual colors on multi-stained slides (37). Three random images were chosen to estimate Pearson’s coefficient values (20× magnification). Pearson correlation coefficient calculated the linear correlation between two variables X and Y.

Western blot, enzyme-linked immunosorbent assay (ELISA) and PCR

Cells were harvested and solubilized in RIPA buffer as we previously described (38). Whole cell lysates (20 μg) were separated by electrophoresis on 8–12% SDS polyacrylamide gels, transferred to PVDF membranes (#10600023, GE Health), and probed with different dilutions of antibodies of interest. The antibodies used in this study, lumican (#ab98067, ab168348) and α-SMA (#ab5694) were purchased from Abcam. Phospho-Smad2 (Ser465/467, #3101), phospho-Smad3 (Ser423/425, #9520), phospho-AKT (Ser473, #4060), phospho-MEK1/2 (Ser217/221, #9154), SMAD4 (#9515), Smad2/3 (#5678), and MEK1/2 (#4694) were purchased from Cell Signaling Technology. Vinculin (#ab18058) and TBP (#ab818) for protein loading control were from Abcam. Reactive bands were visualized with enhanced chemiluminescent reagents (GE Healthcare). For TGF-β stimulation and phosphorylation studies in HPSC, fresh 5% FBS medium was added and the cells were incubated with or without TGF-β (5 ng/ml) for 2, 8, or 24 hours. HPSC nuclear and cytosol lysates were separated by buffer A and buffer C.

The ELISA was performed in fresh supernatant derived from serum-free media of HPSC, HPSC/shSMAD4, HPSC/LUM/KDs, and PDAC cells of 80% confluence in 10cm dishes, washing dishes with PBS twice, and added 3 ml serum-free media per dish with or without treatments. Supernatant was collected at 8 and 24 hours, and ELISA was performed in 5 to 10 X dilutions of the supernatant (#MBS701117, MyBioSource). The optical density of each well was read at 450 nm using a FluoStar Omega reader (BMG Labtech, Inc.).

Total RNA was extracted using TRIzol reagent, and complementary DNA was prepared with the iScript reverse transcription supermix kit (#170-8841, Bio-Rad). The lumican primers forward: 5′-CAGACTGCCTTCTGGTCTCC-3′, and reverse: 5′-AGCTCAACCAGGGATGACAC-3′. The expression level of human LUM mRNA was quantified using iQ SYBR Green Supermix (#170-8880, Bio-Rad). Relative expression levels were determined by normalizing the expression level of each target to GAPDH, and relative mRNA fold changes were determined using the 2 (−ΔΔCt) methods.

SMAD4 and LUM Knockdown Assays in HPSC

Human retroviral short hairpin RNAi against human SMAD4 plasmid (shSMAD4) was purchased from Addgene (plasmid #15724). Three human lumican knockdown shLUM plasmids (shLUM/KDs) were purchased from Sigma-Aldrich (TRC# TRCN0000156246 labeled as shLUM/KD-1, TRCN0000154276 labeled as shLUM/KD-2, TRCN0000153890 as shLUM/KD-3). Detailed products and iRNA sequences are available on Sigma-Aldrich website. Its control non-coding shRNA plasmid was purchased from MD Anderson Core. Procedures for transfection and infection into HPSC were as described previously (38). The effectiveness of SMAD4 knockdown was validated by western blotting analysis. HPSC/shLUM/KD-1, -2, -3, and /CTL were transiently infected in HPSC for 48 hours, and then collections of cell lysate and supernatant for the western blot and ELISA were pursued separately. Simultaneously, adhesion and migration assays were performed. The effectiveness of lumican knockdown was validated by both western blotting and ELISA assays.

Electrophoretic Mobility Shift Assay (EMSA) and LUM promoter luciferase reporter and mutagenesis assays

The LUM promoter fragment was examined for recognition by nuclear proteins by EMSA according to a protocol previously described (38). Three oligo probes containing SBE consensus binding sites were designed and are described in Supplementary Table 1. The super-shift experiments were performed with anti-SMAD4 antibody (#SC-7966×, Santa Cruz). The three SBEs and mutant oligos were purchased from Sigma-Aldrich.

LUM promoter dual-luciferase activity was performed with a pGL2-LUM promoter construct, including part of the LUM first exon and potential SBE sites (SBE1) as previously described (38). The pGL2-LUM plasmid was co-transfected into HPSC and 293T cells with an internal control, TK-Renilla luciferase. The activities of Firefly and Renilla luciferase were determined using a dual luciferase reporter assay system (#E4550, Promega) after 48 hours of transfection. Firefly luciferase activity was normalized to the Renilla luciferase activity of the internal control. For site-directed mutagenesis assays, one base pair in the first SBE sequence in the LUM promoter region from −811 bp to −817 bp was changed using a mutagenesis kit (#200522, Agilent Technologies, Inc.).

Cell matrix adhesion and migration assays

Cell matrix adhesion and migration assays were performed on collagen I coated plates as described (39) (#A11428, 96 well plate. #A11428, 6 well plate. Life Technologies). Briefly, HPSC, HPSC/shSMAD4 cells, and other PDAC cells were seeded (5 × 104 cells) with serum-free medium in the 96 wells where these cells were incubated with or without TGF-β (10 ng/ml), rLUM (0.2μg/ml), supernatant collected from HPSC, or serum free media (as control). HPSC/CTL and HPSC/shLUM/KDs were incubated in serum free media after 48 hours infection. The cells were incubated at 37 °C for 24 hours to determine adhesion to collagen I. Each plate was shaken at 1,000 rpm for 15 seconds and washed twice with PBS carefully to remove non-adherent cells. Afterward, fresh 10% FBS media and 20 μl MTT (5 mg/ml) (#M5655, Sigma-Aldrich) were added into each well subsequently. The plates were incubated for another 3 hours, and the relative number of attached cells was determined by the MTT method.

Time-lapse migration analysis was performed on Olympus IX-81 DSU Spinning Disk Confocal microscope (Olympus) system equipped with the WeatherStation incubator (PrecisionControl) using a 10× objective. Migration was assessed using two-well culture-inserts (#160255, ibidi USA Inc.) placed into 6-well collagen I coated plates. Briefly, these cells were seeded separately into the inserts (3X104) and cultured overnight, and inserts were carefully removed, cells were incubated in serum-free media for 24 hours with or without TGF-β (10 ng/ml) or rLUM (0.2 μg/ml), or HPSC/CTL and HPSC/shLUM/KDs in regular media to allow them to migrate and close the wound. Appropriate channels were recorded for 24 hours by Olympus IX-81 DSU microscope and serial images were saved through SlideBook software. Three random images were chosen for measurement at 0 and 12 hours (10× magnification). The migration rate was determined as the ratio of distance of the wound’s gap at 12 hours versus at 0 hours.

Patient Data

Clinicopathologic and survival data was retrieved from an IRB approved database for the 27 patients (23 patients have complete fellow-up) whose tumors were evaluated by IHC. The lumican expression was scored according to DAB intensity which was quantified using VAQPIS analysis in which the mean value was determined. The patients were then grouped according to DAB staining intensity greater (Stromal lumican high; n= 12) or lower (Stromal lumican low; n = 11) than the mean value. The median survival data and other clinicopathologic parameters between the two groups were compared.

Statistical analysis

All quantified data were plotted and analyzed using GraphPad Prism 6.0. Significance was determined using Student’s unpaired t-test or two-way ANOVA. Data are representative of at least 3 independent experiments and are reported as the mean ± SEM of replicates or triplicates unless otherwise indicated. A P value of <0.05 was considered statistically significant.

Results

1. Expression and secretion of Lumican from PDAC and stromal cells and its effect on cell adhesion and migration

In previous work, we identified that cancer-related survival in pancreatic cancer patients is associated with the amount and distribution of lumican within the primary PDAC tumor (19). In this study we performed IHC staining of a unique cohort of PDAC tumor specimens and observed the presence of lumican within both cancer cells as well as the stroma (Figure 1a). Lumican expression in 27 PDAC TMA samples was then quantitatively analyzed by measuring DAB intensity through the Vectra Automated Quantitative Pathology Imaging System (VAQPIS) (Supplementary figure 1a and Materials and Methods). The results showed lumican expression in stroma was significantly higher than that of the cancer cell compartment, total DAB mean in stroma equals 343,639 (Figure 1b). Double immunofluorescence staining (IF) of the same TMA confirmed the expected physical co-localization of lumican and collagen I with the observation of a statistically significant association (p<0.05) by Prizm analysis (Supplementary figure 1b). Similar methods were applied to test co-localization of lumican and α-smooth muscle actin (α-SMA), a described marker of PSC marker, in the same collection of primary PDAC tumor specimens. Lumican (green) and α-SMA (red) expression levels were positively correlated (Pearson coefficient, r=0.78) indicating that lumican expression in stroma is positively associated with PSC (p>0.0001) (Figure 1c). Using the Kaplan-Meier method, the median survival of the stromal lumican high group (≥DAB mean) was 16.05 months compared to 10.2 months for the stromal lumican low patient group (<DAB mean) (Figure 1d). This trend is consistent with our previous observations (19) although the difference did not reach statistically significance (p=0.32) due to the small sample size. The clinicopathologic characteristics in the patient groups with high or low stromal lumican expression was also compared and is displayed in Supplementary Table 2.

Figure 1. Lumican expression in 27 PDAC specimens in TMA.

Figure 1

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(a) Represented immunohistochemistry images of primary tumors, 10 × (top panel) 40 × (button panel) magnification. (b) VECTRA analysis calculates mean DAB among tumor and stromal cells each case, mean values were plotted by GraphPad Prism. (c) Represented double immunofluorescence staining of lumican (Green) and α-SMA (Red) (left panel), and separate signals per case were recorded and analyzed by FluoView, values were plotted by GraphPad Prism (right). Two TMA cores images (10×) per specimen were taken for analysis. Results are the mean ± SEM. (d) Using the Kaplan-Meier method, overall survival was compared between the patients with primary PDAC tumors with stromal lumican expression levels greater (stromal lumican high; n=12) or less than (stromal lumican low; n=11) the mean signal value.

To confirm our observation from PDAC specimens, we verified lumican expression in pancreatic normal and cancerous cell lines. Expression was analyzed by western blot in immortalized pancreatic ductal cells (HPNE) as well as human pancreatic fibroblasts (HPF) and activated pancreatic stellate cells (HPSC) isolated from human PDAC tumors (32). Pancreatic cancer cell lines (PANC-1, MiaPaCa2, BxPc3, HPAF-II, MDA-PATC43, MDA-PATC50, MDA-PATC53, and MDA-PATC66) were also tested. Lumican was identified higher expression in all three non-cancerous pancreatic cell lines and the PDAC cell lines PANC-1, MiaPaCa2, BxPc3, and MDA-PATC53 (Figure 2a). Lumican mRNA levels were correlated with respective protein levels by RT-PCR (Figure 2b). HPSC, PANC-1, MDA-PATC53, and MiaPaCa2 demonstrated high transcript and protein levels of lumican were therefore selected for further study. To quantify secreted lumican levels, ELISA was performed in serum-free supernatant from these cells at 8 and 24 hours. The results showed that HPSC secreted significantly higher amounts of lumican compared to cancer cells, and among these cancer cells, higher lumican secretion was found in MDA-PATC53 (Figure 2c). HPSC secreted high levels of lumican at both 8 and 24 hours, quantified at approximately 40 ng and 200 ng/1X106 cells/ml, respectively (p<0.0001). MDA-PATC53 cells secreted less lumican, approximately 8 ng and 30 ng/1X106 cells/ml at 8 and 24 hours, respectively (p<0.05) (Supplementary table 3). To confirm the lumican secretion majority in pancreatic stellate cells, results from HPaSteC showed that lumican secretion was exceedingly increased from 8 to 24 hours (5.30 ± 0.48 to 25.29 ± 0.51 ng/1X106 cells/ml, p<0.001) (Supplementary figure 2a and Materials and Methods).

Figure 2. Lumican expression and secretion in pancreatic normal and tumor cell lines, and its secretion level associated with cell adhesion and migration in HPSC and PDAC cells.

Figure 2

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(a) Western blot shows lumican protein in pancreatic cell lines. (b) RT-PCR results show lumican mRNA level in pancreatic cell lines. (c) ELISA results present secreted lumican in HPSC, PANC-1, MDA-PATC53, and MiaPaCa-2 cells at 8 and 24 hours (Absorbance readings were normalized by 1×106 cells/ml). Lumican standard was performed accordingly. Absorbance reading values were plotted by GraphPad Prism, Y= normalized absorbance readings. (d) Adhesion and (e) migration assays were performed onto collagen I coated plates in HPSC, PANC-1, MDA-PATC53, and MiaPaCa2 cells. Results are the mean ± SEM of 3 independent experiments. *p<0.05, ***p<0.001****p< 0.0001.

Recent work suggests ECM acts as a restraining influence within PDAC tumors (32) and our previous study suggested stromal lumican in primary PDAC is associated with longer overall survival and recurrence-free survival (30). Therefore, we hypothesized that the secreted lumican may affect adhesion and migration of cancer cells or HPSC themselves. The cell lines having higher lumican expression and secretion were selected for evaluating their interactions with extracellular matrix. We found adhesion and migration of these cell lines (HPSC and MDA-PATC53) were significantly promoted compared to the cells having lower lumican expression and secretion (Figure 2d and 2e). Cell adhesion or migration are critical step in tumor invasion and metastasis, the process is involved in numerous cellular and extracellular proteins(40), here we were exploring how lumican regulates cell-ECM adhesion and migration in HPSC or PDAC.

2. TGF-β inhibits lumican expression and secretion through canonical signaling pathway in PSC

TGF-β is a potent regulator of cell-matrix and cell-cell adhesion(41), and TGF-β cytokines originate from various cell types within PDAC (42). We therefore sought to understand the relationship between TGF-β and lumican expression in HPSC. We first confirmed that HPSC secrete all three isoforms of TGF-β (TGF-β1, TGF-β2, and TGF-β3), and that TGF-β1 is dominant (Supplementary figure 1c and Materials and Methods). When HPSC were exposed to exogenous TGF-β (5ng/ml) in vitro, the TGF-β pathway was initiated and lumican levels within the cytosol were profoundly reduced after 24 hours, as demonstrated by immunoblotting (Figure 3a). Secreted lumican was also reduced at 8 and 24 hours compared with non-TGF-β exposed cells (p<0.05 and 0.0001, respectively) (Figure 3b). Lumican (Green) and SMAD4 (Red) IF staining after TGF-β exposure confirmed SMAD4 accumulation in the nucleus after 2 hours, and reduction of cytoplasmic lumican after 8 hours (Figure 3c). Similar TGF-β stimulation assays were performed in HPaSteC (Supplementary Materials and Methods), lumican secretion was inhibited significantly when HPaSteC exposed to TGF-β for 8 and 24 hours (p>0.05 or p>0.0001 comparing to no TGF-β exposure, separately) (Supplementary figure 2a), western blotting confirmed lumican expression was reduced and correlated with TGF-β signaling pathway activation after exposing to TGF-β for 24 hours (Supplementary figure 2b).

Figure 3. Lumican expression and secretion reduction while TGF-β pathway activated in HPSC cells.

Figure 3

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(a) Western blot analysis demonstrated expression levels of lumican, SMAD4, phosphor-Smad2, phosphor-Smad3 in nucleus and cytosol of HPSC cells after exposing with or without TGF-β (5 ng/ml) at 2, 8, and 24 hours. Smad2/3 as phosphor-Smad2, or –Smad3 controls. TBP and vinculin were used as the nucleus and cytosol loading control, separately. (b) Secreted lumican level was measured by ELISA after TGF-β treatment (5 ng/ml) or none in 8 and 24 hours in HPSC cells supernatants, lumican readings at 450 nm were normalized by cell number (1 × 106 cells), bar graph was plotted by GraphPad Prism, *P < 0.05 and ****P < 0.0001. (c) Double Immunofluorescence staining of lumican (Green) and SMAD4 (Red) in HPSC cells after exposing to TGF-β at 0, 2, 8, and 24 hours. Nuclei were counterstained with blue DAPI. Images were captured and merged by using an Olympus FV1000 Laser Scanning Confocal microscope and analyzed with FlowView software.

To verify that secreted lumican reduction was a consequence of TGF-β pathway signaling, HPSC were exposed to TGF-β with or without the TGF-β inhibitor, SB431542 (#ab120163, Abcam) for 2 hours (43). In HPSC exposed to TGF-β, the canonical pathway was activated, and cytoplasmic lumican expression reduced. However in the presence of the TGF-β inhibitor, this pathway was inactive and cytoplasmic lumican remained at basal levels. TGF-β non-canonical pathway signaling proteins, such as phospho-AKT and phospho-p44/42, were also not initiated in HPSC (Figure 4a). Lumican (Green) and SMAD4 (Red) IF staining confirmed these findings (Figure 4b). ELISA was performed with the same treatment groups for 24 hours, and demonstrated that secreted lumican was significantly reduced after TGF-β exposure (p<0.05) compared to the combination of TGF-β and SB431542 (p<0.0001) (Figure 4c). After SMAD4 was silenced in HPSC (HPSC/shSMAD4) (Figure 4d), secreted lumican increased significantly at 8 and 24 hours when compared to control HPSC (p<0.0001)(Figure 4e). Together, these results suggest that canonical TGF-β-SMAD4 is a crucial signaling pathway to negatively regulate lumican expression and secretion within pancreatic stellate cells.

Figure 4. Lumican expression and secretion modification after TGF-β pathway blocked or SMAD4 knocked down in HPSC cells.

Figure 4

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(a) Western blot analysis showed lumican, SMAD4, phosphor-Smad2, phosphor-Smad3, phosphor-AKT, and phosphor-p44/42 in nucleus and cytosol of HPSC cells after exposing with or without TGF-β (5 ng/ml) and SB431542 (10 μM) for 2 hours treatment. Smad2/3 as phosphor-Smad2, or –Smad3 controls, AKT and p44/42 as phosphor-AKT and –p44/42 controls. TBP and vinculin were used as the nucleus and cytosol loading control, separately. (b) Double Immunofluorescence staining of lumican (Green) and SMAD4 (Red) in HPSC cells after treated with TGF-β, SB431542, or combination at 2 hours exposure. Nuclei were counterstained with blue DAPI. Images were captured and merged by an Olympus FV1000 Laser Scanning Confocal microscope and analyzed with FlowView. (c) Secreted lumican level was measured by ELISA after exposing to TGF-β (5 ng/ml), SB431542 (10 μM), or combination or none in 24 hours in HPSC cells supernatants. (d) Efficacy of SMAD4 knock-down in HPSC cells was confirmed by western blot. (f) Comparison of secreted lumican level was measured by ELISA in HPSC and HPSC/shSMAD4 cells supernatants at 8 and 24 hours. Bar graphs were plotted by GraphPad Prism, *p<0.05 and ****P < 0.0001.

3. SMAD4 binds to SBEs located within the promoter region of lumican gene (LUM)

Based on our preliminary observations (Figures 3 and 4), we identified three candidate SBE sequences located within the LUM promoter (−811, −3526, and −4303 bp). We hypothesized that these sites cooperate to regulate LUM transcription (Figure 5a). Binding activities of SMAD4 to these sites was determined by EMSA and LUM dual-luciferase reporter assays. EMSA demonstrated that SBE1 oligos have specific binding to HPSC nuclear protein (Lane 1). This binding was only blocked by wild type oligos (Lane 2) and not by mutant oligos (Lane 3), and this was super-shifted by an anti-SMAD4 antibody (Lane 4, arrow showed shift band) (Figure 5b). When HPSC were exposed to TGF-β, SBE1 binding was temporarily decreased at 2 hours, then recovered binding at 8 and 24 hours, though not with other SBEs (Figure 5c). So, a construct including SBE1 was then cloned into pGL2-Basic vectors (Promega) (pGL2-SBE1) to validate the functional activity of specific binding within the LUM promoter (Figure 5a). HPSC and 293T cells were then transiently transfected with pGL2-SBE1 and exposed to TGF-β; luciferase activity was increased 2 hours after TGF-β stimulation suggesting that SBE1 cooperates to enhance LUM activations in both HPSC and 293T cells. Luciferase activity was maintained at both 8 and 24 hours in HPSC (p<0.01 and p<0.0001, respectively), and declined significantly after 8 hours in 293T cells (Figure 5d). To prove the importance of the SBE1 on construct pGL2-SBE1, we specifically mutated the construct at −811 from CAGACA to CACACA (Figure 5e). After this mutagenesis, no luciferase activity in response to TGF-β demonstrating that binding at SBE1 is necessary for LUM transcriptional control (Figure 5f). Together, these assays confirmed that exposure to TGF-β induces SMAD4 translocation and binding to a novel, specific, and functionally active SBE within the LUM promoter.

Figure 5. Map of multiple SBEs in LUM promoter.

Figure 5

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(a) Three SBEs with a CAGACA sequence (blue circles 1, 2, and 3), correspondently to SBEs. (b) EMSA results showed that SBE1 oligos had strong DNA and nuclear protein interaction bands (lane 1), binding was quenched by wild-type (WT) oligos (lane 2) and not by mutant (M) oligos (lane 3), and anti-SMAD4 antibody (Ab) was supershifted binding activity (lane 4). (c) EMSA shows SBE1 binding activities were regulated by exposing to TGF-β at 2, 8, and 24 hours, but not on SBE3. OCT-1 DNA binding activities were determined as loading controls. (d) The SBE1 sequence in the LUM promoter (Top) and the Mut1 mutant sequence (G→C) (Bottom). (e) LUM pGL2-SBE1 promoter dual-luciferase activity construct of potential SBE1 site (double arrow on 1a) was stimulated by TGF-β (5ng/ml), the ratios of pGL2-LUM activities were presented at 0, 2, 8, and 24 hours in HPSC and 293T cells. (f) LUM pGL2-MUT1 Luciferase activities ratios of Mut1 with or without TGF-β (5ng/ml) at 0, 2, 8, and 24 hours in HPSC and 293T cells. Results are the mean ± SEM of 3 independent experiments. **p<0.01 and ****P < 0.0001.

4. Lumican regulation affects HPSC adhesion and migration in vitro

As previously experiments demonstrated that higher lumican-secreting cells, HPSC and MDA-PATC53, achieved increased adhesion and enhanced migration, versus the lower lumican-secreting cells PANC-1 and MiaPaCa2, and correlation between lumican and collagen I was also confirmed in these patients’ specimen lumican/collagen I double IF staining which showed Pearson r=0.47 (p>0.05) (Supplementary figure 1b). More assays were performed to further confirm how lumican regulation was specifically involved in these adhesion and migration phenomena in vitro. An anti-lumican antibody was used to deplete lumican in HPSC supernatant for 24 hours, which resulted in less secreted lumican on ELISA compared with HPSC treated with an IgG1 control antibody (p<0.001) (Figure 6a). Correspondingly, HPSC adhesion was decreased in the antibody treated group compared to serum-free or IgG1 treated groups (p<0.05) (Figure 6b). In HPSC with silenced SMAD4 (HPSC/shSMAD4), adhesion to collagen I was increased (Figure 6c). Migration assays demonstrated increased motility in HPSC exposed to rLUM (p<0.0001) as well as in HPSC/shSMAD4 (p<0.05), but not in TGF-β treatment group, which indicates TGF-β likely impact HPSC motility. These results clearly demonstrates that external or secreted lumican promote HPSC motility in vitro (Figure 6d). More specific assays to validate the correlation of lumican expression or secretion with HPSC’s motility were performed. While silencing lumican in HPSC by three lumican shRNA plasmids, HPSC transiently was infected by the three plasmids (shLUM/KD-1, shLUM/KD-2, and shLUM/KD-3). Effectiveness of lumican knock down was validated by both western blotting (Figure 6e) and ELISA (Figure 6f) comparing with HPSC/CTL, and adhesion of these lumican knock down clones significantly decreased (p<0.01 or p<0.001) (Figure 6g), correspondingly with migration significantly delayed in these clones (p<0.001 or p<0.01 comparing to control) (Figure 6h). Representative images of migration in HPSC, HPSC treated with TGF-β, HPSC treated with rLUM, and HPSC/shSMAD4 at 6 and 12 hours were shown in supplementary figure 3a, and representative images in HPSC/CTL and three HPSC/shLUM/KDs at 6 and 12 hours were shown in supplementary figure 3b as well. These data demonstrate that lumican augments HPSC adhesion and migration in a collagen I-rich environment.

Figure 6. Lumican secretion associated with cell matrix adhesion and migration in collagen I coated plates.

Figure 6

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(a) ELISA shows secreted lumican was reduced after HPSC cells supernatant were treated with specific lumican antibody (b) Secreted lumican reduction correlated with decreased HPSC adhesion. (c) Cell matrix adhesion in HPSC and HPSC/shSMAD4 cells. (d) Time lapse of migration assays were performed on according of HPSC, HPSC plus TGF-β, HPSC plus rLUM, and HPSC/shSMAD4 conditions in collagen I coated plates, separately. (e) Western blotting showed effectiveness of lumican knock down in HPSC/CTL and HPSC/shLUM/KDs. (f) ELISA showed secreted lumican reduction in three HPSC/shLUM/KDs’ clones comparing with HPSC/CTL (p<0.01 and p<0.001). (g) Adhesion assays showed reduction in these HPSC/shLUM/KDs clones comparing to HPSC/CTL (p<0.01 and p<0.001). (h) Time lapse of migration assays showed migration delayed in these HPSC/shLUM/KDs clones comparing to HPSC/CTL (p<0.001 and p<0.01). Bar graphs were plotted by GraphPad Prism. Results are the mean ± SEM. *p<0.05, **p<0.01, and ****P < 0.0001.

Intriguingly, supernatant from HPSC can also increase the adherence of low-secreting lumican PDAC cell lines, such as PANC-1 and MiaPaCa2 cells, to collagen I (p<0.0001) (Supplementary figure 4a). Conversely, HPSC supernatant depleted of lumican by anti-lumican antibody suppresses PANC-1 adhesion (p<0.01) (Supplementary figure 4b), while supernatant from HPSC/shSMAD4 enhances PANC-1 adhesion (p<0.01) (Supplementary figure 4c). Panc-1 cell adhesion was reduced (p<0.01) after exposure to supernatant obtained from HPSC after exposure to TGF-β and reduction in lumican production (Supplementary figure 4d). We co-cultured HPSC and PANC-1 (where HPSC were labeled with GFP and PANC-1 labeled with RFP), and serial images were analyzed at several time points. The results showed that HPSC migrated faster and circulated near PANC-1 cells (Red arrows) (Supplementary figure 4e). Together, these results suggest that HPSC secreted lumican influences the adhesion and motility of both tumor and stromal cells in a collagen I rich environment.

Discussion

In this study, we found that activated pancreatic stellate cells derived from human primary PDAC tumors secrete large amounts of lumican; a finding which correlates with the co-localization of extracellular lumican with α-SMA expressing PSC, and fibrillar collagen, in primary tumors. We demonstrated that TGF-β uses its canonical pathway to negatively regulate lumican production in HPSC; this occurs through by SMAD4 translocation and binding to a SBE (CAGACA) within the promoter region of the lumican gene. Interestingly, we found that secreted lumican in the extracellular environment preferentially enhances cell adhesion and migration of HPSC when on collagen. Together, these data provide a clearer understanding of the control mechanisms and consequences of extracellular lumican within PDAC tumors.

Lumican is usually localized to areas of pathologic fibrosis and has been demonstrated in the ECM within various human malignancies including pancreatic cancer (8–10, 44, 45). In pancreatic cancer, lumican mRNA is highly expressed in acinar cells, islet cells and proliferating fibroblasts within the desmoplastic stroma surrounding cancer cells and activated stellate cells have been implicated as the cell source (9). Similar studies have demonstrated that PSC are the cell source of fibrillar collagen within the desmoplastic ECM of PDAC tumors (20). The co-localization of lumican with desmoplasia is logical given our understanding that lumican binds collagen fibrils and regulates interfibrillar spacing during collagen assembly (17, 18, 46). TGF-β is one of the strongest inducers of ECM production during fibrogenesis (24, 25), and largely drives the desmoplastic stroma observed in pancreatic carcinoma (26). TGF-β mediates Type I collagen gene expression through a synergistic cooperation of the transcription factors Smad2/Smad3 and Sp1 acting on the promoter (24, 47). To date, however, no studies have described the mechanism by which TGF-β could control lumican production by stellate cells activated within PDAC tumors.

In the described experiments we demonstrate that TGF-β acts through its canonical signaling pathway to negatively control lumican transcription in PSC through novel SMAD4-SBE binding. Additionally, we demonstrate how extracellular lumican influences the function of PSC within a collagen rich environment. ELISA of media conditioned by PSC (HPSC and HPaSteC) and other cell types within PDAC allowed us to demonstrate that PSC, activated stellate cells harvested from primary PDAC, secrete large amounts of lumican into the extracellular space. Manipulation of the TGF-β canonical signaling pathway allowed us to determine that negative regulation at the transcriptional level is the primary means by which the cytokine TGF-β exerts control over lumican secretion from PSC. These observations expand and provide needed detail to previous reports (9).

Several studies have demonstrated that SLRP proteins, specifically decorin, can modulate cellular behavior, including cell migration and proliferation during embryonic development, tissue repair, and tumor growth, in addition to their extracellular matrix function as regulators of tissue hydration and collagen fibrillogenesis (48–50). We previously found that the presence of lumican in the ECM surrounding was associated with improved patient outcome after multimodal therapy and surgical removal of localized PDAC (39) suggesting that lumican influences cells within the tumor microenvironment. Our laboratory and others have demonstrated that extracellular lumican impacts the activity and survival of pancreatic cancer cells (39, 40), but the effects of extracellular lumican on host cells is poorly characterized. Our observations show that high levels of lumican secretion augment adhesion of HPSC and PDAC cancer cells to collagen I, and this effect is abrogated by anti-lumican antibodies and genetic silencing of lumican in HPSC. This suggests that extracellular lumican, regardless of the cell source, effects cell adhesion of multiple cell types in a collagen-rich environment. Interestingly, lumican also increased cell migration when on collagen I, but the migration of HPSC was increased to greater degree than cancer cells. The molecular mechanism of these phenotypic differences remains to be elucidated, but the observations serve as an example of the differential influence of lumican on the cellular compartment.

These findings offer a deeper understanding of the biological function of lumican within the PDAC tumor-microenvironment, and provide a fundamental platform upon which to discover other predictive markers or to design novel anti-tumor strategies in PDAC patients.

Supplementary Material

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Translational Relevance.

The tumor microenvironment, in particular the tumor-stroma interaction, is an important factor of disease progression and outcome in patients with pancreatic ductal adenocarcinoma (PDAC). Lumican is an extracellular matrix protein whose presence with primary PDAC tumors is associated with improved patient outcome. Here we identify that activated pancreatic stellate cells isolated from primary PDAC tumors secrete large amounts of lumican that augments adhesion and migration of activated pancreatic stellate cells in a collagen-rich environment. We found that transforming growth factor-β, a cytokine associated with desmoplasia in PDAC, acts through its canonical signaling pathway to inhibit lumican transcription and secretion in activated stellate cells. Together, these findings underscore the influential role of lumican within the stroma of localized PDAC and its impact on tumor biology and patient outcome.

Acknowledgments

Funding Support: Viragh Family Foundation and Various Donors in Pancreatic Cancer Research (to JBF), and National Institutes of Health grant T32CA009599 (to DR and MVRP).

Grants Support

The study was supported by Cancer Center Support Grant (CCSG) core resources of Flow Cytometry and Cellular Imaging Facility under NIH/NCI award (P30CA016672). Sequencing and Microarray Facility is funded by NCI #CA016672. CCSG for the STR DNA fingerprinting and the Characterized Cell Line cores were funded by NCI grant (CA016672). Drs. D. Roife and M.V. Rios Perez were supported in part by National Institutes of Health (NIH) grant T32CA009599.

Footnotes

Disclosure of Potential Conflicts of Interest: The authors have no potential conflicts of interest to declare.

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