Elevated oxidative stress is an aberration seen in many solid tumors, and exploiting this biochemical difference has the potential to enhance the efficacy of anticancer agents. Homeostasis of reactive oxygen species (ROS) is important for normal cell function, but excessive production of ROS can result in cellular toxicity, and therefore ROS levels must be balanced finely. Here, we highlight the relationship between the extracellular matrix and ROS production by reporting a novel function of the matricellular protein Fibulin-5 (Fbln5). We used genetically engineered mouse models of pancreatic ductal adenocarcinoma (PDAC) and found that mutation of the integrin-binding domain of Fbln5 led to decreased tumor growth, increased survival, and enhanced chemoresponse to standard PDAC therapies. Through mechanistic investigations, we found that improved survival was due to increased levels of oxidative stress in Fbln5-mutant tumors. Furthermore, loss of the Fbln5–integrin interaction augmented fibronectin signaling, driving integrin-induced ROS production in a 5-lipooxygenase–dependent manner. These data indicate that Fbln5 promotes PDAC progression by functioning as a molecular rheostat that modulates cell–ECM interactions to reduce ROS production, and thus tip the balance in favor of tumor cell survival and treatment-refractory disease. Cancer Res; 75(23); 5058–69. ©2015 AACR.
Tumors develop and progress in the context of the extracellular matrix (ECM). In fact, structural ECM proteins can promote tumor cell survival and stimulate invasive tumor cell programs (1–6). This is particularly evident in desmoplastic tumors, such as pancreatic ductal adenocarcinoma (PDAC; 7), where ECM proteins, including fibronectin and collagen, activate signaling pathways that drive cell survival, proliferation, and migration (1, 2). ECM-mediated signaling is governed by expression of the ECM proteins, the presence of cell surface receptors and the expression and activity of matricellular proteins that function as extracellular adaptors to reduce ECM–cell interaction (1, 8, 9). Fibulin-5 (Fbln5), a member of the fibulin family of proteins, is particularly important in this regard, as it binds to α4β1 and α5β1 integrins via an RGD sequence, but does not support integrin activation (8). Thus, Fbln5 competes with structural ECM ligands, principally fibronectin, that would otherwise activate these integrins.
ECM proteins stimulate the generation of reactive oxygen species (ROS) in an integrin-dependent manner (2, 10, 11). ROS generation in this context is generally transient and serves primarily as a signaling intermediate that enhances cellular activity (12). We reasoned that a chronic increase in integrin-induced ROS would negatively affect tumor growth. Fibronectin-driven ROS generation is an attractive pathway to exploit for this strategy because fibronectin ligation of β1 integrins is governed in part by Fbln5 (8, 13). We reported previously that Fbln5 reduced fibronectin-mediated integrin-induced ROS production by competing with fibronectin for binding to α5β1 integrin (13). Mutation of the three amino acid RGD sequence in Fbln5 to RGE abolishes integrin binding yet preserves other functions of Fbln5 (14, 15). An essential function of Fbln5 is elastic fiber formation (16). As a result, Fbln5−/− mice exhibit disorganized elastic fibers throughout the body, leading to tortuous great vessels, emphysematous lungs, and loose skin, resembling cutis laxa syndrome in humans (17, 18). In contrast, Fbln5RGE/RGE (RGE) mice have intact elastic fibers and are essentially indistinguishable from wild-type (WT) littermates (15). However, RGE mice show increased levels of ROS compared with WT animals in tissues where fibronectin is abundant (15). Given the high expression of fibronectin in the stroma of PDAC and increasing evidence supporting enhanced ROS production as an anticancer strategy (19), we evaluated the consequence of ablating the integrin-binding ability of Fbln5 in robust spontaneous models of PDAC. Our results show that Fbln5–integrin interaction promotes aggressive tumor growth and progression in mice and that 5-lipooxygenase (5-Lox) activity was required for ROS induction in the absence of functional Fbln5. In addition, we found that Fbln5 was expressed abundantly in the stroma of human PDAC tumors. These data provide insight into the function of Fbln5 in PDAC and reveal how ECM signaling might be exploited to drive pro-oxidant therapy.
Materials and Methods
Fbln5RGE/RGE (RGE), Fbln5−/− (KO), LSL-KrasG12D/+; Cdkn2aLox/Lox (KI) and Cdkn2aLox/Lox; p48Cre (IC) mice were generated as previously described (1, 15, 20, 21). RGE mice were used to breed with KI and IC mice to generate genetically matched LSL-KrasG12D/+; Cdkn2aLox/Lox; p48Cre (KIC) mice and RGE-KIC mice. LSL-Trp53R172H/+ mice were obtained from National Cancer Institute (NCI) Mouse Repository (22). RGE mice were also used to breed with LSL-KrasG12D/+; LSL-Trp53R172H/+ (KP) and p48Cre mice to generate genetically matched LSL-KrasG12D/+; LSL-Trp53R172H/+; p48Cre (KPC) mice and RGE-KPC mice. All mice were housed in a pathogen-free facility and all experiments were performed under written protocols approved by the Institutional Animal Care and Use Committee at the University of Texas Southwestern Medical Center at Dallas.
For endpoint studies, KIC and RGE-KIC mice were sacrificed and entire tissues, including pancreas/tumor, liver, and spleen, were harvested and weighed at 1, 1.5, and 2-months-old. KPC and RGE-KPC mice were sacrificed at 3 and 5 months, n ≥ 8 mice per time point per group. For all survival studies, mice were carefully monitored and sacrificed when they appeared moribund. For antioxidant treatment, N-acetyl cysteine (NAC; Sigma-Aldrich) was given to mice at 7 mg/mL in the drinking water from 4-weeks-old until moribund. For endpoint therapy experiments, KIC and RGE-KIC mice were treated for 3 weeks starting at 7 weeks (1.5 months) of age with i.p. injection of low dose Gemcitabine (GemL; 12.5 mg/kg 3×/week) or Abraxane (Abx; 5 mg/kg 2×/week). Mice were sacrificed and tissues were isolated for analysis, n ≥ 6 mice per group. For survival studies with therapy, cohorts of KIC and RGE-KIC mice were treated similarly with GemL, high dose Gemcitabine (GemH; 50 mg/kg 1×/week i.p.) or Abx until moribund.
Histology, immunohistochemistry, and immunofluorescence staining
Tissues were snap frozen and embedded in OCT (Tissue-Tek) for frozen sections or fixed with 4% paraformaldehyde overnight and embedded in paraffin for sectioning. Frozen sections were fixed in ice-cold acetone for 5 minutes, air dried for 10 minutes followed by 10 minutes incubation with PBS to dissolve the OCT. Paraffin sections were deparaffinized and rehydrated with xylene and serial dilutions of ethanol followed by antigen retrieval with 0.01 mol/L citric acid buffer (pH 6.0). Sections were blocked with 20% aquablock and incubated with primary antibodies in blocking solution (5% BSA in TBST) at 4°C overnight. Primary antibodies used for frozen sections were: rabbit polyclonal anti-mouse Fbln5 (1:100; purified polyclonal IgG by our lab, 1.6 mg/mL; ref. 17), rat anti-Meca32 (1:100; purified IgG from hybridoma by our laboratory; 1.0 mg/mL; ref. 23), goat anti-Amylase (1:2,000; sc-12821; Santa Cruz Biotechnology), rabbit anti-fibronectin (1:100; DP3060; Acris), and rabbit anti-γH2AX (1:50; NB100-2280; NOVUS). Primary antibodies used for paraffin sections were: rabbit anti-human Fbln5 (1:75; HPA000868; Sigma-Aldrich), rabbit anti-Phospho-Histone H3 (PH3; 1:100; 06-570, Millipore), rabbit anti-Amylase (1:2,000; 3796S; Cell Signaling Technology), and rat anti-endomucin (1:100; sc-65495, Santa Cruz Biotechnology). Fluorescein isothiocyanate (FITC)–conjugated donkey anti-rabbit, rat IgGs, Cyanine 3 (Cy3)–conjugated donkey anti-rat, mouse, rabbit IgGs and horseradish peroxidase–conjugated donkey anti-rabbit IgGs from Jackson ImmunoResearch were used as secondary antibodies.
Slides with sections of FFPE de-identified human pancreatic cancer tissue were obtained from the UT Southwestern Tissue Resource and the Department of Pathology, UT MD Anderson Cancer Center. Human PDAC sections were stained for Fbln5 expression using rabbit anti-Fbln5 (HPA000868; Sigma-Aldrich) as indicated above.
Western blot analysis
Western blots were performed as previously described (24). In brief, protein lysates from cell culture or tumor tissues were extracted in ice-cold RIPA buffer (50 mmol/L Tris-Cl, 150 mmol/L NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) containing cocktails of protease (Thermo Fisher Scientific) and phosphatase inhibitors (Sigma-Aldrich), by centrifugation (13,000 × g, 15 minutes) at 4°C following three freeze-thaw cycles. Proteins were separated by SDS-PAGE and transferred to methanol-activated polyvinylidene difluoride membrane (VWR). The primary antibodies used include the following: rabbit anti-mouse Fbln5 (1:1,000), rabbit anti-human Fbln5 (1:500; HPA000868; Sigma-Aldrich), anti-Nqo1 (1:1,000; ab34173; Abcam), anti–α-tubulin (1:1,000; ab4047; Abcam) and anti–β-actin (1:5,000; A2066; Sigma-Aldrich). Horseradish peroxidase–conjugated donkey anti-rabbit IgG (1:10,000; Jackson Immunoresearch) as secondary antibodies were used.
Cell lines used include mouse endothelial cell (EC) line bEnd.3 (13), mouse pancreatic cancer cell lines Pan02 (13) and mPLR B9 (1), and human pancreatic cell lines MiaPaCa-2, AsPC-1, and Panc1 (all purchased from the ATCC). NG2+ cells were isolated using anti-NG2 antibody–conjugated magnetic beads from a KIC tumor (25). Fbln5+/+ (WT), Fbln5−/− (KO) and Fbln5RGE/RGE (RGE) mouse embryonic fibroblasts (MEF) were isolated from embryonic day E12.5-E14.5 embryos and genotypes were confirmed by PCR. bEnd.3 cells were treated with 100 μmol/L H2O2 or 10 μg/mL α5β1 integrin–activating antibody and lysates were collected for Western blot analysis. MEFs were cultured in reduced serum medium Opti-MEM (Life Technologies) overnight before being plated on plastic, fibronectin (Sigma-Aldrich) or collagen I (Thermo Fisher Scientific), each at 10 μg/mL unless otherwise noted. After plating, MEFs were grown in serum-free medium supplemented with fibronectin, collagen, β1 integrin blocking antibody (each at 10 μg/mL) or various chemicals. Inhibitors used for various ROS sources include Rotenone (R8875-1G; Sigma Aldrich), Diphenyleneiodonium chloride (DPI; D2926, Sigma-Aldrich) and nordihydroguaiaretic acid (NDGA; 479975; Millipore). All cells were maintained in DMEM (Mediatech, Inc.) with 10% FBS and were grown in 37°C humidified incubator with 5% CO2. MEFs were used between passages 2–5 for all experiments.
The detailed protocol on ROS detection and quantification has been described previously (13). In brief, for tissues, 5 μmol/L dihydroethidium (DHE; D11347; Life Technologies) was applied to freshly sectioned tissues and incubated at 37°C for 30 minutes. 6–10 images were taken randomly from each tissue and at least three tissues were included in each group. Fluorescence intensity was quantified by the software NIS-Elements. To visualize ROS in cells, 10 μmol/L of 2′-7′-dicholordihydrofluorescein diacetate (DCF-DA; D-399, Life Technologies) was added to cells grown on fibronectin-coated chamber slides. 8–10 pictures were taken randomly from each condition and area fraction was quantified and normalized to cell number by DAPI using the software NIS-Elements. Three independent experiments for each condition were evaluated.
qPCR array and real-time PCR
WT and RGE MEFs were cultured in reduced serum medium Opti-MEM overnight before plating on fibronectin. Upon plating, MEFs were grown in serum-free medium supplemented with fibronectin for 4, 16, and 24 hours. RNA lysates were isolated using the RNeasy Plus Mini Kit (Qiagen). The RT2 First Strand Kit (Qiagen) was used for cDNA synthesis. Then cDNA samples were subjected to RT2 Profiler PCR array to analyze gene-expression changes related to mouse oxidative stress and antioxidant defense pathways (Qiagen; PAMM-065A). Experiments were performed and data were analyzed following the manufacturer's instructions. All the candidate genes were further checked and confirmed by real-time PCR. Ribosome protein S6 (RPS6) was used as the internal control. Following primers were used for real-time PCR: Nqo1 (forward): 5′-AGACCTGGTGATATTTCAGTTCCCATTG-3′; Nqo1 (reverse): 5′- CAAGGTCTTCTTATTCTGGAAAGGACCGT-3′; RPS6 (forward): 5′-AAGCTCCGCACCTTCTAT-3′; RPS6R (reverse): 5′-TGACTGGACTCAGACTTAGAAGTAGAAGC-3′.
Nqo1 activity assay
Nqo1 enzyme activity was measured in a reaction mixture containing 200 μmol/L NADH (Sigma-Aldrich) as an electron donor and 10 μmol/L menadione (Sigma-Aldrich) as an Nqo1 substrate and intermediate electron acceptor as described (26, 27). Cytochrome c serves as the terminal electron acceptor. Therefore, the measured rate of cytochrome c reduction correlates with Nqo1 enzymatic activity. To prepare lysates, cells were scraped in PBS and samples were sonicated. Lysate was added to the reaction mixture and the reduction of cytochrome c (Sigma-Aldrich) over two minutes was monitored by absorbance at 550 nm. Dicoumarol, a selective inhibitor of Nqo1, was added as a negative control. Enzyme activity units were calculated as nmol of cytochrome c reduced/min/μg lysate.
For statistical analysis, the unpaired t test was used for comparison between genotypes and various groups. The log-rank (Mantel–Cox) test was used for all the mouse survival studies. Overall, P value less than 0.05 was considered as statistically significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.
Fbln5 expression in pancreatic cancer
The expression of Fbln5 is prominent in developing arteries and is diminished in most adult organs, but can be reactivated in injured vessels (14). We examined Fbln5 expression in multiple mouse and human cell lines and tumor lysates. The mouse EC line bEnd.3 and fibroblasts, including MEFs and fibroblasts isolated from mouse PDAC (NG2+ cells), expressed Fbln5 (Fig. 1A and Supplementary Fig. S1B) as did human and mouse PDAC lysates (Fig. 1B and Supplementary Fig. S1C). In contrast, pancreatic cancer cell lines, including three human (MiaPaCa-2, Panc1, and AsPC-1; Fig. 1B) and four mouse lines (Pan02 and three isogenic lines isolated from mouse PDAC), did not express detectable levels of Fbln5 protein (Fig. 1A, Supplementary Fig. S1B and data not shown). However, all cell lines and PDAC tumor lysates examined express α5β1 integrin (Supplementary Fig. S1A and S1C), which serves as the cell surface receptor for Fbln5 and fibronectin (8).
Fbln5 IHC in syngenic pancreatic Pan02 tumors grown subcutaneously in Fbln5+/+ (WT) or Fbln5−/− (KO) mice revealed that Fbln5 is produced by stromal cells (Fig. 1E). Costaining of Fbln5 with the EC marker Meca32 shows that Fbln5 can be produced by ECs within the tumor (Fig. 1E). IHC analysis of Fbln5 expression in multiple mouse models of PDAC revealed Fbln5 reactivity mainly in the stroma (Fig. 1C and D and data not shown). We also examined FBLN5 in human PDAC by IHC and found the protein was expressed in all human PDACs examined (n = 25). The staining pattern was confined largely to the stromal compartment; however, not all regions of stroma were positive for FBLN5 protein (Fig. 1F). The nature of the heterogeneous stromal staining pattern is unclear but suggests that FBLN5 expression is controlled tightly.
Characterization of KIC and KPC mice
To evaluate the contribution of Fbln5 to PDAC development and progression, we used KIC and KPC mice, two well established conditional genetically engineered mouse models (GEMM) of PDAC based on the p48Cre (also known as Ptf1a) driver, which is expressed in pancreatic bud progenitor cells (22, 28,29). KIC animals express an active form of Kras and have biallelic inactivation of the Cdkn2a locus (LSL-KrasG12D/+; Cdkn2aLox/Lox; p48Cre; ref. 29). KPC animals express the same activating G12D mutation in Kras and also harbor a R172H point mutation in p53, the Li-Fraumeni human ortholog (LSL-KrasG12D/+; LSL-Trp53R172H/+; p48Cre; ref. 22). Histologic examination of KIC and KPC pancreatic tissue by a pathologist revealed that each model developed early pancreatic intraepithelial neoplasias (PanINs) and highly infiltrative adenocarcinomas ranging from well-differentiated areas with clear malignant gland formation to areas that were more poorly differentiated (Supplementary Fig. S2A). Similar to human PDAC, Masson's trichrome staining showed extensive collagen deposition in the area of PanINs and PDAC in KIC and KPC tumors (Supplementary Fig. S2B).
Ablation of Fbln5–integrin interaction reduces tumor growth and prolongs survival
Mutation of the Fbln5–integrin-binding sequence from RGD to RGE renders the protein incapable of binding to integrins (14). Fbln5RGE/RGE (RGE) mice are viable, fertile and phenotypically normal compared with WT animals (15). To study the contribution of Fbln5 to PDAC development, we crossed RGE mice with KIC or KPC animals to generate genetically matched KIC and RGE-KIC, KPC and RGE-KPC mice. There was no difference in Fbln5 expression levels between KIC and RGE-KIC or KPC and RGE-KPC tumors (Fig. 1G and Supplementary Fig. S1D). However, tumors had significantly increased Fbln5 expression compared with normal pancreas (Fig. 1C and G and Supplementary Fig. S1D). Pancreatic and mouse body weights were determined in KIC and RGE-KIC mice at 1, 1.5, and 2 months (Supplementary Fig. S2C and 2A). There is no significant difference for tumor/body weight at 1 and 1.5 months (Supplementary Fig. S2C). RGE-KIC mice exhibited significantly lower tumor/body weight at 2 months than KIC mice, with tumor weights ranging from 0.24 to 0.86 gram for RGE-KIC mice and 0.72 to 1.11 gram for KIC mice (Fig. 2A). This is consistent with significantly reduced proliferating cells in RGE-KIC tumors (Fig. 2C and D). Similar results were observed in the KPC model, which were analyzed at 3 and 5 months of age (Fig. 2B, E, and F).
Survival analysis revealed that RGE-KIC mice lived significantly longer than KIC mice (Fig. 2G) with a median survival of 74 days for RGE-KIC mice and 61.5 days for KIC mice. KIC and RGE-KIC mice appeared normal with no obvious phenotype up to 1.5 months of age but later became moribund, usually accompanied by weight loss. Moreover, some mice developed jaundice or ascites. Autopsies revealed the presence of large solid tumors with limited gross metastases. Liver micrometastasis was seen in the majority of KIC and RGE-KIC mice in the survival study necropsies (Supplementary Fig. S2D). Similarly, RGE-KPC show significantly prolonged survival compared with KPC mice (175 vs. 143.5 days; Fig. 2H). KPC and RGE-KPC mice appeared healthy up to 3-month-old and were sacrificed between 3 and 9 months, commonly presenting with body weight loss, jaundice, or ascites. Gross liver metastasis was seen in 30% to 40% of animals (Supplementary Fig. S2E and S2F).
Increased oxidative stress in RGE-KIC and RGE-KPC tumors
We reported previously that the growth of subcutaneous Pan02 tumors in Fbln5−/− mice was significantly reduced compared with WT animals due to increased ROS (13). Dihydroethidium (DHE) staining of tumors from the GEMMs showed that the Fbln5 RGE mutation significantly induced ROS levels in KIC and KPC tumors (Fig. 3A and B). Accordingly, the level of γH2AX, a commonly used marker for oxidative stress-induced DNA damage (30), was higher in RGE-KIC tumors than KIC tumors (Fig. 3C). However, in the context of normal pancreatic tissue, the Fbln5 RGE mutation did not alter ROS levels (Supplementary Fig. S3A and S3B). This is consistent with the level of fibronectin expression, which was elevated in PDAC compared with normal pancreatic tissue (Fig. 1H). To determine whether ROS induction contributed to the prolonged survival in RGE animals, KIC and RGE-KIC mice were treated with the antioxidant NAC and survival was examined (Fig. 3D). Prolonged NAC treatment decreased survival of RGE-KIC mice but did not affect the survival of KIC mice (Fig. 3D). ROS production was also examined in NAC-treated KIC and RGE-KIC tumors, which revealed no difference between the two groups (Supplementary Fig. S3C and S3D). Collectively, ROS induction driven by the Fbln5 RGE mutation resulted in reduced tumor growth and prolonged survival.
Angiogenesis is reduced in RGE-KIC and RGE-KPC tumors
Prior studies indicate that Fbln5 can modulate angiogenesis (31), and we reported that loss of Fbln5 resulted in decreased angiogenesis in pancreatic tumors (13). Therefore, we examined microvessel density (MVD) in KIC and RGE-KIC, KPC, and RGE-KPC tumors by co-immunostaining with the EC marker endomucin and the acinar cell marker amylase. MVD was significantly reduced in RGE-KIC compared with KIC in tumor regions as marked by loss of amylase reactivity (Fig. 3E and F and Supplementary Fig. S4A). Immunostaining and quantification of MVD from 3- to 5-month-old KPC and RGE-KPC tumors also revealed significantly reduced MVD in RGE-KPC tumors (Fig. 3G and H and Supplementary Fig. S4B and S4C). We also examined the MVD of KIC and RGE-KIC tissues at 1-month-old. At this time point, more than 90% of the tissue retained amylase expression. There was no difference in MVD in amylase-positive areas between KIC and RGE-KIC tissues (Supplementary Fig. S5A–S5C). In addition, pancreas tissue from nontumor-bearing WT and RGE mice were analyzed for MVD (Supplementary Fig. S5D). Again, no significant difference between the two groups was observed (Supplementary Fig. S5E and S5F). We found that the number of proliferating ECs costained with phospho-histone H3 and endomucin was decreased in tumors in RGE animals compared with tumors in WT mice, supporting the reduction of MVD in RGE-KIC and RGE-KPC tumors (Fig. 3I–M and Supplementary Fig. S4D and S4E). Overall, the reduction of MVD correlated with tumor-specific induction of ROS in RGE animals (Fig. 3A and Supplementary Fig. S4A, 3B and Supplementary Fig. S4B and S4C). These data suggest that the absence of functional Fbln5 impairs EC survival specifically in the tumor microenvironment.
Induction of the oxidative stress–responsive gene Nqo1 by fibronectin-induced ROS in vitro and in vivo
Our data suggested that Fbln5 controls ROS production through fibronectin–β1 integrin interaction. To elucidate the underlying molecular mechanism of ROS generation, we isolated WT, KO, and RGE primary MEFs. Fbln5 expression levels were equivalent between WT and RGE MEFs (Supplementary Fig. S6). We found that ROS was elevated in KO and RGE MEFs but not in WT MEFs when cells were plated on fibronectin (Fig. 4A). Next, we performed qPCR arrays to screen for oxidative stress and antioxidant response pathway–related genes using RNA harvested from WT and RGE MEFs. From these arrays, NADP(H):quinone oxidoreductase 1 (Nqo1) was a reproducible and reliable target that was increased in RGE MEFs after plating on fibronectin. Nqo1 is an antioxidant enzyme that is responsible for the reduction of quinones to hydroquinones using NAD(P)H as an electron donor (32). Reducing quinone levels lowers the occurrence of ROS generation as a result of redox cycling (32). Induction of Nqo1 can be mediated by the Keap1–Nrf2–ARE pathway (33). The induction of Nqo1 in RGE MEFs when plated on fibronectin was confirmed by quantitative real-time PCR (Fig. 4B), enzymatic activity (Fig. 4C), and Western blotting (Fig. 4D). Concordantly, the induction of Nqo1 expression was reversed by antioxidant treatment (with NAC) in a dose-dependent manner (Fig. 4E), showing that the elevation of Nqo1 is a consequence of increased ROS status in RGE MEFs. In addition, Nqo1 induction was elevated in tumors from RGE animals (Fig. 4F and G).
Nqo1 induction is dependent on fibronectin–β1 integrin interaction and 5-Lox activity
It has been reported that integrin activation by fibronectin can induce ROS production (12). Accordingly, when the EC line bEnd.3 was treated with a α5β1 integrin-activating antibody, Nqo1 levels were induced (Fig. 5A). The induction of Nqo1 was specific to activation by fibronectin and was not present when cells were plated on plastic or collagen (Fig. 5B). Induction was partially blocked by β1 integrin blockade (Fig. 5B). Given this data, we conclude that the induction of Nqo1 is responsive to ROS production induced by fibronectin-mediated β1 integrin ligation.
To determine the cellular source of ROS production in the absence of Fbln5–integrin interaction, we used inhibitors for various ROS sources, including the mitochondrial respiratory chain inhibitor Rotenone, NADPH oxidase (NOX) inhibitor diphenyleneiodonium chloride (DPI), and 5-Lox inhibitor, nordihydroguaiaretic acid (NDGA). Treatment with Rotenone or DPI did not suppress the induction of Nqo1, suggesting that mitochondria and NOX are not the intracellular source of ROS production (Fig. 5C). In addition, there was no induction of NOX enzymatic activity in RGE MEFs compared with WT MEFs by fibronectin (Supplementary Fig. S7). In contrast, inhibition of 5-Lox by NDGA reduced Nqo1 levels, indicating 5-Lox as the potential source of ROS (Fig. 5D and E). This is consistent with a previous discovery that 5-Lox contributes to a strong burst of ROS production by fibronectin–integrin engagement (11).
ROS induction has an additive therapeutic effect when combined with standard chemotherapy agents
To determine whether increased integrin-induced ROS improved response to chemotherapy, we compared the efficacy of standard chemotherapy agents Gemcitabine (Gem) and Abraxane (Abx) in KIC and RGE-KIC mice. All therapy started at 1.5 month old when KIC and RGE-KIC mice had established solid tumors (Supplementary Fig. S2C). We found that low-dose Gemcitabine (GemL) and Abx were more effective in the context of mutant Fbln5 (Fig. 6A and B). Survival studies were also performed with cohorts of KIC and RGE-KIC mice treated with GemL, high-dose Gemcitabine (GemH) and Abx. RGE-KIC mice consistently survived significantly longer in all three treatment groups than similarly treated KIC mice (Fig. 6C–E). These data suggest that increasing integrin-induced ROS augments the activity of standard chemotherapy.
We demonstrated that Fbln5 expression is induced in a significant percentage of pancreatic cancers and that it promotes tumor progression by competing with fibronectin for integrin ligation. Global loss of Fbln5–integrin interaction resulted in decreased tumor growth and prolonged survival of tumor-bearing mice with no apparent adverse effects in normal tissues. The decrease in tumor burden was dependent on increased fibronectin-mediated integrin activation, which increased ROS production through 5-Lox activity and resulted in reduced angiogenesis in the tumor microenvironment. These findings are summarized in Fig. 7.
The ECM provides a structural framework in which tumors develop and progress. ECM signaling contributes to cell survival, proliferation, and migration; thus, regulation of cellular events initiated by the ECM is critical for tumor progression. To date, pharmacologic modification of the ECM in PDAC has not resulted in improvement of chemoresponse or overall survival in patients (34–36). However, preclinical studies focused on inhibiting pathways that stimulate ECM deposition (e.g., TGFβ) have shown promise in promoting tumor control (37). Here, we have highlighted that increased integrin activation can result in decreased tumor growth by elevating integrin-induced ROS production. The extent of cell–ECM interaction is regulated in part by matricellular proteins, including Fbln5. Yet, the contribution of Fbln5 to cancer has been limited largely to expression studies (38–41), which have not defined a clear function for Fbln5 in tumorigenesis. We found that FBLN5 protein was expressed in all of the human PDAC samples (n = 25) we evaluated. Expression was largely restricted to the stromal compartment, yet the pattern of expression was not uniform as there were some areas of stroma that were negative or only weakly reactive. This heterogeneity suggests that evaluation of FBLN5 and potentially other matricellular proteins in human tissue microarrays could be challenging. Additional studies on the expression of Fbln5 protein in clinically annotated tumor samples are needed to elucidate whether Fbln5 expression has predictive value.
To extend our studies, we sought to understand how Fbln5–integrin interaction functions in the context of the microenvironment of PDAC. We used two distinct but related GEMMs of PDAC that recapitulate common mutations observed in the human disease (42–44). The expression of Fbln5 in each model is similar to the expression level and pattern of Fbln5 expression in human PDAC. Furthermore, fibronectin and α5β1 integrin are expressed abundantly in animal models of PDAC as well as human PDAC (45, 46). To study Fbln5–integrin interaction, we took advantage of the fact that (i) Fbln5 binds but does not activate α5β1 (8, 14), suggesting that it can function to reduce binding of other ligands of the integrin; and (ii) knockin mice carrying a point mutation in the integrin binding domain of Fbln5 (RGE mice) are viable and fertile (15). The described essential function of Fbln5 is in elastic fiber assembly as shown by Fbln5-deficient animals (17, 18) and biochemical analysis (16, 47). However, RGE mice have intact elastic fibers (15), indicating that Fbln5–integrin binding is not required for elastic fiber assembly. These data strongly suggest that the phenotype in the RGE animals is not due to changes in elastic fiber assembly but a result of an increase in integrin activation by ligands other than Fbln5. Given the dramatic increase in fibronectin expression as well as other stromal components in PDAC, we postulated that the tumor microenvironment would provide a biologically meaningful stress to ascertain the functional consequences of increased integrin ligation in RGE animals.
ROS production as a result of integrin ligation is a well established (11, 12), although underappreciated signaling pathway. Previously, we discovered that the loss of Fbln5–integrin interaction results in increased integrin-induced ROS production (13). Cellular redox homeostasis is tightly regulated by the balance between ROS scavenging and eliminating systems (19). Cancer cells often generate higher levels of ROS due to metabolic abnormality, activation of oncogenes or loss of functional p53 (19). For example, increased levels of ROS, particularly H2O2 are highly mutagenic and contribute to elevated mutation levels and heterogeneity. Thus, an imbalance in ROS scavenging and eliminating systems is likely to result in acute consequences in the tumor microenvironment. For example, increasing ROS levels might result in inhibition of cell proliferation and ultimately cell death (48). However, cancer cells have developed adaptive mechanisms to manage increased ROS levels (19). One adaptive mechanism is the induction of the antioxidant response transcription factor Nrf2 to increase the expression of the ROS detoxification enzyme Nqo1 (49, 50). We found that the loss of Fbln5–integrin interaction induces Nqo1, and that this response is ROS dependent. Nqo1 levels as a result further validated the elevation of oxidative stress in tumors grown in RGE mice and also provided a tractable biochemical endpoint to evaluate the signaling cascade induced by fibronectin in the absence of integrin-binding Fbln5. Furthermore, using Nqo1 levels as an endpoint, we discovered that ablation of Fbln5–integrin interaction increased 5-Lox activity in a fibronectin-dependent manner. Pharmacologic inhibition of 5-Lox rescued the fibronectin-driven phenotype in vitro, implicating that 5-Lox is downstream of integrin activation. This is consistent with previous reports showing that integrin activation by fibronectin can stimulate ROS production primarily through 5-Lox (11, 12). Fibronectin has also been reported to stimulate intracellular ROS in pancreatic cancer cells through NOX and the mitochondria (10), although we did not find evidence of this in our system.
We found that stromal and tumor cells express the integrin profile required for fibronectin-induced ROS production. However, changes in ECs were the most apparent phenotype in tumors from RGE mice. ECs are sensitive to elevated ROS (51) and this was evident by the consistent reduction in MVD and reduction in proliferating ECs in tumors grown in RGE animals. Fibroblasts from RGE mice produce elevated levels of ROS in culture in a fibronectin and integrin-dependent manner. Yet, surprisingly, we found no significant changes in the presence or activation of fibroblasts in tumors from RGE mice (data not shown). Global analysis of ROS using DHE indicates that many cell types, including tumor cells, display elevated ROS levels in tumors from RGE mice. However, in vitro studies suggest that Fbln5 does not affect ECM-mediated ROS induction in tumor cells (data not shown). It is plausible that long-lived ROS molecules (e.g., H2O2) travel from stromal cells and increase oxidative stress in tumor cells, resulting in decreased proliferation and reduced tumor growth. It is also feasible that ECs in the tumor microenvironment succumb to elevated ROS induced by mutant Fbln5 and the decreased tumor growth is akin to an antiangiogenic effect. In contrast, Fbln5 null mice display an exaggerated vascular response after subcutaneous implantation of polyvinyl alcohol sponges (31). However, the mechanism of how Fbln5 directly affects EC function and the contribution of integrins in this phenotype is poorly understood. In our model, nontumor-bearing pancreata of WT and RGE mice show similar MVD (Supplementary Fig. S5). However, in the context of the tumor microenvironment, the basal level of ROS is increased compared with normal pancreas; therefore, inducing further ROS by mutation of Fbln5 may explain the negative effect on EC function. If so, this suggests that the mutation in Fbln5 functions as an endogenous inhibitor of angiogenesis selectively in the tumor microenvironment. These hypotheses are currently being evaluated.
Our studies show that Fbln5 functions as a rheostat to dampen integrin-mediated ROS production. This function of reducing cell–ECM interaction is similar to what has been observed for other matricellular proteins. For example, SPARC reduces the binding of fibrillar collagens to discoidin domain receptors, thereby reducing collagen-induced cell signaling and attachment (1). Current studies are focused on understanding factors that drive Fbln5 expression in the tumor microenvironment and identification of the integrin-mediated signaling pathway that activates 5-Lox in the context of mutant Fbln5. Overall, our study illustrates how the matricellular protein Fbln5 functions to reduce fibronectin–integrin interaction and suggests that Fbln5 is a novel therapeutic target for pancreatic cancer.
Disclosure of Potential Conflicts of Interest
R.A. Brekken reports receiving a commercial research grant from Remeditex Ventures and has ownership interest in patent on Fbln5 as a target for cancer therapy. No potential conflicts of interest were disclosed by the other authors.
The funders had no role in study design, data collection, data analysis, decision to publish, or preparation of the article.
Conception and design: M. Wang, M. Topalovski, R.A. Brekken
Development of methodology: M. Wang, M. Topalovski, Z.R. Moore, D.H. Castrillon
Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): M. Wang, J.E. Toombs, Z.R. Moore, H. Yanagisawa, H. Wang, A. Witkiewicz
Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): M. Wang, Z.R. Moore, H. Wang, A. Witkiewicz, D.H. Castrillon
Writing, review, and/or revision of the manuscript: M. Wang, M. Topalovski, Z.R. Moore, D.A. Boothman, H. Yanagisawa, H. Wang, D.H. Castrillon, R.A. Brekken
Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): M. Wang, M. Topalovski, C.M. Wright, D.A. Boothman
Study supervision: D.A. Boothman, R.A. Brekken
The authors acknowledge helpful discussions with Drs. Michael Dellinger and Adi Gazdar and members of the Brekken laboratory.
This work was supported in part by grants from the American Cancer Society (ACS, RSG-10-244-01-CSM to R.A. Brekken), The Joe and Jessie Crump Medical Research Foundation (R.A. Brekken), NIH (R01 CA118240 to R.A. Brekken; R01 CA137181 to D.H. Castrillon; and T32 GM008203 to M. Topalovski), the Effie Marie Cain Scholarship in Angiogenesis Research (R.A. Brekken), and Remeditex Ventures. The UT Southwestern Tissue Resource is supported by the NCI (5P30 CA142543).
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