Loss of Fibulin-5 Binding to B1 Integrins Inhibits Tumor Growth by Increasing the Level of ROS

Loss of fibulin-5 binding to b1 integrins inhibits tumor growth by increasing the level of ROS

Marie K. Schluterman1, Shelby L. Chapman2, Grzegorz Korpanty1, Kiyoshi Ozumi3, Tohru Fukai3, Hiromi Yanagisawa2 and Rolf A. Brekken1,*

SUMMARY Tumor survival depends in part on the ability of tumor cells to transform the surrounding extracellular matrix (ECM) into an environment conducive to tumor progression. Matricellular proteins are secreted into the ECM and impact signaling pathways that are required for pro-tumorigenic activities such as angiogenesis. Fibulin-5 (Fbln5) is a matricellular protein that was recently shown to regulate angiogenesis; however, its effect on tumor angiogenesis and thus tumor growth is currently unknown. We report that the growth of pancreatic tumors and tumor angiogenesis is suppressed in Fbln5-null (Fbln5–/–) mice compared with wild-type (WT) littermates. Furthermore, we observed an increase in the level of reactive oxygen species (ROS) in tumors grown in Fbln5–/– animals. Increased ROS resulted in elevated DNA damage, increased apoptosis of endothelial cells within the tumor, and represented the underlying cause for the reduction in angiogenesis and tumor growth. In vitro, we identified a novel pathway by which Fbln5 controls ROS production through a mechanism that is dependent on b1 integrins. These results were validated in Fbln5RGE/RGE mice, which harbor a point mutation in the integrin-binding RGD motif of Fbln5, preventing its interaction with integrins. Tumor growth and angiogenesis was reduced in Fbln5RGE/RGE mice, however treatment with an antioxidant rescued angiogenesis and elevated tumor growth to WT levels. These findings introduce a novel function for Fbln5 in the regulation of integrin-induced ROS production and establish a rationale for future studies to examine

DMM whether blocking Fbln5 function could be an effective anti-tumor strategy, alone or in combination with other therapies.

INTRODUCTION contrast, papilloma development is delayed in osteopontin-null The tumor microenvironment contains many components that mice (Hsieh et al., 2006). It is clear from these studies that, with influence tumor survival and progression; namely, activated further investigation, matricellular proteins could be valuable fibroblasts, infiltrating macrophages, neutrophils and endothelial targets for controlling cancer growth and spread (Pozzi et al., 2000; cells that are recruited into the tumor mass and actively remodel Proia and Kuperwasser, 2005). Therefore, we are interested in the extracellular matrix (ECM) (Farrow et al., 2008; Mantovani determining the effect of the matricellular protein Fbln5 on tumor et al., 2008). The acellular component of the tumor growth. microenvironment, the ECM, consists of a three-dimensional Fbln5 is a member of the fibulin family of proteins and consists network of structural proteins such as fibronectin and collagen of six calcium-binding epidermal growth factor-like repeats, one that function as an architectural scaffold for cells, and provides of which contains an integrin-binding RGD motif and a COOH- the context for cell function and tissue development. The terminal fibulin-type module (Timpl et al., 2003; Zheng et al., 2007). organization of, and the interaction between, these components Fbln5 is crucial for proper assembly of elastic fibers as it tethers Disease Models & Mechanisms within the tumor microenvironment are crucial determinants of tropoelastin to microfibrils where it is crosslinked by lysyl oxidase tumor survival. Interactions between cells and the ECM stimulate to form mature elastic fibers (Liu et al., 2004; Zheng et al., 2007). signaling pathways that regulate cellular activities such as survival, Mice that are deficient in Fbln5 have fragmented and disorganized proliferation and migration – processes that are crucial to tumor elastic fibers leading to loose skin, vascular anomalies and alveolar expansion (Albig and Schiemann, 2005; Li et al., 2007). defects (Yanagisawa et al., 2002). Fbln5 protein is secreted into the Matricellular proteins, such as fibulin-5 (Fbln5), associate with ECM by fibroblasts, vascular smooth muscle cells (VSMCs) and the matrix and regulate the crosstalk between cells and their endothelial cells, and associates with cells through integrins aVb3, surrounding ECM. Numerous studies have shown that a4b1 and a5b1 (Lomas et al., 2007; Timpl et al., 2003). Recent studies matricellular proteins can have strong effects on tumor growth. have suggested that Fbln5 can regulate angiogenesis. In vitro For example, tumor progression is generally enhanced in mice experiments showed that Fbln5 antagonized vascular endothelial lacking the matricellular proteins SPARC (secreted protein acidic growth factor (VEGF) signaling thereby abrogating VEGF-induced and rich in cysteine) or thrombospondin-1 or -2 (Brekken et al., proliferation and migration of endothelial cells (Albig et al., 2006; 2003; Lawler and Detmar, 2004; Puolakkainen et al., 2004). By Albig and Schiemann, 2004). In addition, in vivo studies indicated an increase in vascular invasion into polyvinyl sponges that were implanted into Fbln5–/– mice compared with wild-type (WT) 1Hamon Center for Therapeutic Oncology Research and Departments of Surgery animals (Sullivan et al., 2007). The exact mechanism by which Fbln5 and Pharmacology and 2Molecular Biology, University of Texas Southwestern exerts its effect on angiogenesis is unknown. Given these results Medical Center, Dallas, TX 75390, USA Fbln5 3 and the aberrant vessel formation in -null animals, we sought Departments of Medicine and Pharmacology, University of Illinois at Chicago, Fbln5–/– Chicago, IL 60612, USA to determine whether tumor growth is altered in mice *Author for correspondence ([email protected]) owing to effects on angiogenesis.

Disease Models & Mechanisms 333 RESEARCH ARTICLE Decreased tumor growth in Fbln5–/– mice

Herein, we report the first results from tumor studies performed tumors from WT and Fbln5–/– animals revealed no striking in Fbln5–/– mice. Interestingly, the growth of an implanted murine differences in morphology, tissue architecture or fibrillar collagen pancreatic cancer cell line (Pan02) was decreased in mice lacking deposition (supplementary material Fig. S1). As a control, we endogenous Fbln5. Moreover, blood vessel density in the tumor evaluated the expression of Fbln5 in Pan02 cells by reverse microenvironment of Fbln5–/– mice was reduced significantly transcription (RT)-PCR and western blot analysis, and determined compared with tumors grown in WT littermates. This effect was that Pan02 cells do not express Fbln5. This was confirmed by the result of elevated reactive oxygen species (ROS) production immunohistochemistry staining showing that tumors from WT within tumors from Fbln5–/– mice, which increased oxidative animals express high levels of Fbln5, whereas tumors from Fbln5–/– damage and augmented endothelial cell death. This led us to the mice were devoid of any Fbln5 immunoreactivity. These results discovery that, in vitro, Fbln5 regulates ROS production in both indicated that only host cells found within the tumor expressed fibroblasts and endothelial cells through a mechanism that is Fbln5 and not the tumor cells (supplementary material Fig. S1). dependent on b1 integrin binding to fibronectin. We reproduced Importantly, a significant portion of the Fbln5 present in tumors these findings in vivo by performing tumor studies in mice from WT mice was localized to blood vessels and perivascular areas expressing a form of Fbln5 that is deficient in the ability to bind to (supplementary material Fig. S1). integrins (Fbln5RGE/RGE mice). Likewise, we observed a reduction in tumor growth and angiogenesis in these Fbln5RGE/RGE mice, and Proliferation decreased and apoptosis increased in tumors from again observed increased levels of ROS. Finally, when Fbln5RGE/RGE Fbln5–/– mice mice were treated with an antioxidant, blood vessel density A disruption in tumor growth is often caused by a decrease in the increased and tumor growth was restored. These results identify proliferation rate of cells that make up a tumor, along with an a novel function for Fbln5 in the regulation of integrin-induced increase in the death of these cells. To further confirm that tumor ROS production and give us insight in to how Fbln5 affects growth in Fbln5–/– mice was attenuated, we examined these angiogenesis. characteristics in Pan02 tumors from WT and Fbln5–/– animals.

DMM Immunohistochemistry with an antibody that is specific for RESULTS phosphorylated histone H3 revealed a significant decrease in the Pancreatic tumor growth is reduced in Fbln5–/– mice number of proliferating cells in tumors grown in the absence of To evaluate tumor growth in Fbln5–/– mice, Pan02 cells, a murine Fbln5 (Fig. 2). By contrast, TUNEL (TdT-mediated dUTP nick-end pancreatic carcinoma cell line, were injected subcutaneously into labeling) analysis showed a significant increase in the number of WT and Fbln5–/– mice. The subcutaneous tumor volume was apoptotic cells in tumors grown in Fbln5–/– animals compared with recorded weekly throughout the experiment. Tumors grew WT mice (Fig. 2). These results correlate with the anticipated significantly slower in Fbln5–/– mice than tumors in WT littermates characteristics of smaller, slower growing tumors such as those seen (Fig. 1A). After four weeks, the mice were sacrificed and the tumors in Fbln5-deficient animals. weighed. The tumors in Fbln5–/– mice were smaller than the tumors from WT animals (Fig. 1B). This result was recapitulated in our Angiogenesis is decreased in tumors from Fbln5–/– mice pancreatic orthotopic model where Pan02 cells were injected Sustained tumor growth requires angiogenesis to facilitate nutrient directly into the tail of the pancreas (Fig. 1C). Tumors were first delivery to the tumor mass. Given this knowledge and the recent palpable two weeks after tumor cell injection into the pancreas. At studies suggesting that Fbln5 is a regulator of angiogenesis, we four weeks, animals were sacrificed and the pancreas with the evaluated vascular density in tumors from Fbln5–/– mice to tumor was harvested and weighed. The final orthotopic tumor determine whether the decrease in tumor growth was because of Disease Models & Mechanisms weights in Fbln5–/– mice were again smaller when compared with an inadequate blood supply (Sullivan et al., 2007). We examined tumors grown in WT littermates (Fig. 1C). We also observed a the blood vessel density in tumors by immunohistochemistry decrease in the number of peritoneal and lymph node metastases using the endothelial cell marker MECA-32 (Fig. 3A). The number in Fbln5–/– mice (Fig. 1D). Histological analysis of orthotopic of blood vessels per mm2 was significantly lower in subcutaneous

Fig. 1. Pancreatic tumor growth is reduced in Fbln5–/– mice. Murine pancreatic carcinoma cells (Pan02) were injected subcutaneously (3ϫ106 cells) (A,B) or orthotopically (5ϫ105 cells) (C,D) into WT and Fbln5–/– mice. (A) Mean subcutaneous tumor volume ± standard error of the mean (s.e.m.) of a representative experiment (n=5 for WT and Fbln5–/–). (B) Mean final subcutaneous tumor weight ± s.e.m. for three individual experiments (WT, n=18; Fbln5–/–, n=19). (C) The mean tumor/pancreas weight ± s.e.m. and (D) the number of observed metastatic events (Farrow et al., 2008) from WT (n=7) and Fbln5–/– (n=6) animals injected orthotopically with Pan02 cells. *P<0.05, **P<0.01.

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Fig. 2. The absence of host Fbln5 results in increased apoptosis and decreased proliferation in Pan02 tumors. Formalin-fixed sections of subcutaneous tumors from WT (n=4) and Fbln5–/– (n=3) mice were evaluated for the levels of apoptosis by TUNEL staining (TUNEL, top panels) and cell proliferation by immunohistochemistry for phosphorylated histone H3 (Phospho-H3, bottom panels). The number of TUNEL- or phospho-H3-positive cells was determined by fluorescence microscopy using tumor slides counterstained with DAPI. Metamorph imaging software was used to quantify the percentage signal above a threshold, which was set by evaluating sections Fig. 3. Vessel density is decreased in tumors grown in Fbln5–/– mice. that were treated with secondary antibody alone. Ten pictures (total (A) Sections from subcutaneous and orthotopic tumors were stained with magnification, 200ϫ) were taken from each tumor for quantification. The MECA-32 (green), and counterstained with DAPI (blue), to analyze blood vessel DMM results shown are mean±s.e.m. *P<0.01, **P<0.001, Student’s t-test. Bars, density. The numbers of MECA-32-positive structures were counted in a 50 μm. minimum of six microscopic fields (total magnification, 200ϫ) from each tumor (at least three tumors per genotype) in both the subcutaneous (top panels) and orthotopic (bottom panels) settings. *P<0.05, ***P<0.0001, Student’s t-test. Mean blood vessel density ± standard deviation (s.d.). (Fig. 3A, top panels) and orthotopic (bottom panels) tumors grown (B) MECA-32-positive blood vessel density in non-tumor-bearing normal in Fbln5–/– mice compared with WT animals. These results pancreas from WT and Fbln5–/– mice was determined by suggested that the reduced tumor growth observed in Fbln5–/– mice immunohistochemistry. Mean blood vessel density ± s.e.m. is shown. *P<0.05. was caused, in part, by the inability of tumors to develop and Bars, 50 μm. maintain a sufficient blood vessel network. Previous results have reported an increase in vessel sprouting in Fbln5–/– animals (Sullivan et al., 2007). Therefore, we next asked increase in ROS in tumors grown in Fbln5–/– mice compared with whether the reduction in angiogenesis that we observed was tumors from WT mice (Fig. 4A). However, the expression and specific to the tumor microenvironment. To answer this, we activity of SOD3 was not decreased in tumors from Fbln5–/– mice analyzed blood vessel density in the pancreas of non-tumor- compared with WT mice (data not shown), suggesting that the bearing WT and Fbln5–/– animals. Consistent with results accumulation of ROS is not because of a loss of SOD3 expression. Disease Models & Mechanisms described previously, we observed an increase in the number of It is well known that, owing to the rapid metabolic rate of tumor vessels in the pancreas of non-tumor-bearing Fbln5–/– animals cells, the tumor microenvironment exists in a high oxidative state compared with WT animals (Fig. 3B). These results indicated that containing elevated levels of ROS compared with normal tissue the reduction in blood vessels observed in tumors grown in (Das, 2002). Knowing this, we hypothesized that the additional Fbln5–/– mice was due to a characteristic solely of the tumor increase in ROS due to the loss of Fbln5 would lead to a state of microenvironment and not present under non-pathological chronic oxidative stress within the tumor microenvironment. conditions. Chronic oxidative stress would result in more oxidative damage; therefore, we analyzed the level of two markers of oxidative Increased ROS level in tumors grown in Fbln5–/– mice damage, 8-hydroxydeoxyguanosine (8-OHdG) and phosphorylated We next sought a mechanism for the specific reduction of histone H2A (gH2AX) (Tanaka et al., 2006; Valavanidis et al., 2009). angiogenesis in the tumor microenvironment. Recently, Fbln5 has Correlating with the increase in ROS, we observed a significant been shown to interact with superoxide dismutase 3 (SOD3), a increase in the level of 8-OHdG and gH2AX in tumors grown in protein that is important in the breakdown of superoxides, a Fbln5–/– mice compared with tumors from WT animals (Fig. 4B,C). common ROS (Nguyen et al., 2004). It was reported that, in aortas Low levels of ROS are well tolerated by endothelial cells; however, of Fbln5–/– mice, the loss of Fbln5 reduced the amount of available chronic oxidative stress induces endothelial cell dysfunction and SOD3 and caused a build-up of ROS within the aorta. Therefore, death (Touyz and Schiffrin, 2004). To determine whether the we hypothesized that tumors grown in Fbln5–/– mice would also elevated production of ROS in tumors from Fbln5–/– mice was have an increase in the level of ROS. To test this, we examined affecting endothelial cell survival, we examined the amount of ROS levels in tumors from WT and Fbln5–/– mice. endothelial cell death in tumors from WT and Fbln5–/– animals by Dihydroethidium (DHE)-based analysis revealed a significant analysis of cleaved caspase 3. We observed a significant increase

Disease Models & Mechanisms 335 RESEARCH ARTICLE Decreased tumor growth in Fbln5–/– mice DMM

Fig. 4. Oxidative stress is elevated in tumors grown in Fbln5–/– mice. (A) Fresh frozen sections of subcutaneous Pan02 tumors from WT (n=3) and Fbln5–/– (n=3) animals were incubated with dihydroethidium (DHE) for in situ detection of ROS. The resulting DHE-mediated fluorescence was quantified by Metamorph software. A representative image from tumors grown in WT and Fbln5–/– animals is shown. The results shown are the mean fluorescence ϫ intensity (±s.e.m.) from at least five 200 fields from each tumor. (B,C) The Fig. 5. Fbln5–/– mouse embryonic fibroblasts produced higher levels of presence of DNA damage in frozen sections of subcutaneous Pan02 tumors ROS in a fibronectin- and b1 integrin-dependent fashion. MEFs harvested from WT ( 5) and –/– ( 7) animals was detected by n= Fbln5 n= from WT and Fbln5–/– mice were grown on fibronectin or collagen. (A,B) After immunohistochemistry with an antibody specific for 8-OHdG (B) and H2AX g 24 hours, the cells were stained with DCF-DA (green) to detect ROS. Fbln5–/– (C) followed by development with a fluorescently conjugated secondary MEFs plated on fibronectin expressed higher levels of ROS than WT MEFs antibody. A representative image from tumors grown in WT and –/– Fbln5 grown on fibronectin or Fbln5–/– MEFs grown on collagen. ROS production by animals is shown. The results shown are the mean fluorescence intensity Fbln5–/– MEFs was inhibited by the addition of recombinant full-length Fbln5 ϫ (±s.e.m.) from at least five 400 fields from each tumor stained. (D) Pan02 (rFbln5), but not by mutant Fbln5 where the RGD domain had been deleted. Disease Models & Mechanisms –/– tumors from WT (n=3) and Fbln5 (n=3) mice were sectioned and stained DCF-DA staining was quantified by normalizing to cell number by DAPI with MECA-32 (green) for blood vessel identification and co-localized with staining (blue). Ten pictures (total magnification, 200ϫ) were taken from each anti-cleaved caspase 3 antibody (red) to determine apoptotic endothelial cells. condition and used for analysis. The assay was performed in triplicate. *P<0.05. The total number of dying endothelial cells was determined by hand counting (C) ROS production was reduced by blocking b1 activation, but was not and graphed as a percentage of the total number of apoptotic cells. affected by a control antibody (IgG). The level of ROS was determined by Arrowheads indicate double positive cells. Ten pictures were taken from each incubating with DCF-DA. Ten pictures (total magnification, 200ϫ) were taken ϫ tumor section (total magnification, 200 ) for analysis. Results shown are of each condition and used for analysis. DCF-DA staining was normalized to ±s.e.m. *P<0.05, **P<0.01. Bars, 50 μm. cell number by DAPI staining. The assay was performed in triplicate. The results shown are mean±s.e.m. *P<0.05. Bars, 50 μm.

in the number of apoptotic endothelial cells in tumors from Fbln5–/– mice compared with WT animals (Fig. 4D). Therefore, we proposed that the decrease in blood vessel density observed in (MEFs) harvested from Fbln5–/– and WT embryos. Infiltrating tumors from Fbln5–/– mice was due, in part, to increased oxidative fibroblasts make up a large percentage of the cells found in the stress and damage on endothelial cells making them unable to tumor microenvironment, therefore MEFs provided us with a survive in the tumor microenvironment. means of initially investigating the effect of Fbln5 on ROS production in a tumor-relevant cell population (Kunz-Schughart Fbln5 controls fibronectin-mediated integrin-induced ROS and Knuechel, 2002). MEFs were grown on fibronectin- or collagen- production coated chamber slides or 96-well plates, and the level of ROS was To further validate the effect of Fbln5 on ROS production, we evaluated by DCF-DA staining. Fbln5–/– MEFs produced performed in vitro studies using mouse embryonic fibroblasts significantly higher levels of ROS than WT MEFs when plated on

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fibronectin but not on collagen or gelatin (Fig. 5A,B; data not shown). Thus, cell association with fibronectin in the absence of Fbln5 was necessary for elevated ROS formation. To determine whether the difference in ROS production was due directly to a loss of Fbln5 expression, Fbln5–/– MEFs were treated with full- length recombinant Fbln5 protein prior to plating on fibronectin. Treatment with full-length Fbln5, but not with an NH2-terminal truncation mutant that lacks the RGD-integrin binding motif, reduced ROS production by Fbln5–/– MEFs (Fig. 5B). This result indicated that Fbln5 binding to integrins was necessary to block ROS generation. The a5b1 integrin is the primary fibronectin receptor and it has been shown that binding of this integrin to fibronectin can stimulate downstream signaling, leading to ROS generation (Chiarugi et al., 2003). To rule out an elevation in b1 integrin expression as a cause of the increased ROS seen in null MEFs, we examined the expression of b1 integrins by RT-PCR and observed equal –/– expression between WT and Fbln5 MEFs (data not shown). Fig. 6. Fbln5 controls integrin-induced ROS production in endothelial Lomas et al. (Lomas et al., 2007) recently provided evidence that cells. bEnd.3 cells were transfected with siRNAs to reduce expression of Fbln5. Fbln5 competed with fibronectin for b1 integrin binding. (A) As determined by western blot, transfection with two siRNAs directed Furthermore, it was also shown that binding of Fbln5 to b1 against Fbln5 reduced its expression, whereas transfection with a siRNA designed from a scrambled sequence (SCR) did not reduce Fbln5 expression. integrins does not cause activation of the integrin or of downstream UNT, untreated. b-actin was used as a loading control. (B) Twenty-four hours DMM signaling pathways (Lomas et al., 2007). To determine whether the after transfection, cells were plated on fibronectin-coated 96-well plates and effect on ROS formation was dependent on activation of b1 stained with DCF-DA for ROS detection. Fluorescence was measured on a plate –/– integrins, we treated Fbln5 and WT MEFs, plated on fibronectin, reader. Treatment with both Fbln5 siRNAs increased ROS production, whereas with a b1 function-blocking antibody. Addition of the antibody antioxidant (NAC) treatment reduced ROS expression. (C) The assay was reduced the level of ROS produced by Fbln5–/– MEFs, whereas the repeated by plating cells on either fibronectin or collagen. Only cells plated on control IgG antibody had no effect (Fig. 5C). These results suggested fibronectin had a significant increase in ROS production, whereas treatment that Fbln5 competes with fibronectin for integrin binding and, in with an anti-b1 integrin function-blocking antibody inhibited this increase. Results shown are mean±s.e.m. **P<0.001, *P<0.05. the absence of Fbln5, b1 integrin activation by fibronectin is increased leading to higher levels of ROS. Results from our tumor studies suggested that the higher level of ROS present within tumors from Fbln5–/– mice affected on Pan02 ROS production. As stated previously, we showed that endothelial cell survival. Therefore, we next wanted to determine Pan02 cells do not express Fbln5; therefore, we determined the whether Fbln5 controlled ROS generation in endothelial cells by a effect of exogenous Fbln5 on Pan02 cells. Before plating on mechanism similar to that observed in fibroblasts. We treated fibronectin-coated 96-well plates, tumor cells were treated with a bEnd.3 cells, a mouse endothelial cell line shown to express b1 full-length recombinant Fbln5 protein. After one hour, cells were integrins (Sheibani and Frazier, 1998), with small interfering RNA treated with DCF-DA to measure the level of ROS. Although plating Disease Models & Mechanisms (siRNA) to knockdown the expression of Fbln5. We confirmed, by on fibronectin induced an increase in ROS, Fbln5 did not inhibit western blot, that two siRNAs directed against Fbln5 had production as seen in the MEFs and endothelial cells, indicating successfully reduced the expression in bEnd.3 cells (Fig. 6A). Fbln5 that ROS production by tumor cells was not affected by the loss expression was not affected by transfection of a scrambled siRNA. of host Fbln5 (supplementary material Fig. S2). After transfection, cells were plated on either fibronectin- or collagen-coated 96-well plates and stained with DCF-DA. The loss Tumor growth is reduced and ROS levels are increased in of Fbln5 expression significantly increased ROS formation in Fbln5RGE/RGE mice endothelial cells plated on fibronectin but not on collagen; this was We determined that the binding of Fbln5 to integrins is crucial for reversed with the addition of the antioxidant N-acetyl cysteine its regulation of ROS production. To confirm this in vivo, we (NAC) (Fig. 6B,C). Treatment with the b1 integrin function- repeated our tumor studies in transgenic knock-in mice containing blocking antibody inhibited the increase in ROS production within an altered RGD motif. In these mice, the aspartic acid of the these cells (Fig. 6C). These results confirmed that Fbln5 regulates integrin-binding RGD sequence of Fbln5 was changed to glutamic integrin-induced ROS production in endothelial cells, and provided acid, rendering the protein unable to bind to integrins (Yang et al., further support that one possible mechanism for the reduced 2007). These mice appear normal and do not exhibit any of the angiogenesis in tumors from Fbln5–/– mice was increased ROS defects observed in Fbln5–/– mice (H. Yanagisawa, unpublished). generation by endothelial cells. This increase, combined with the Pan02 cells were injected subcutaneously into WT, Fbln5–/– and high level of ROS already present within the tumor, would apply Fbln5RGE/RGE mice, and tumor growth was monitored. As observed chronic oxidative stress to endothelial cells resulting in cell death. in the Fbln5–/– mice, tumors from Fbln5RGE/RGE mice grew slower Finally, to determine whether the loss of Fbln5 caused tumor and were smaller than tumors from WT animals (Fig. 7A,D). At cells to increase production of ROS, we analyzed the effect of Fbln5 the time of sacrifice, tumors were harvested and the level of ROS

Disease Models & Mechanisms 337 RESEARCH ARTICLE Decreased tumor growth in Fbln5–/– mice

Fig. 7. Tumor growth and angiogenesis is reduced in Fbln5RGE/RGE mice, but restored with antioxidant treatment. (A-C) Pan02 cells were injected subcutaneously into WT (n=8), Fbln5–/– (n=7) and Fbln5RGE/RGE mice (n=8). The final mean (±s.e.m.) tumor weight at the time of harvest, 4 weeks post-tumor cell injection, is displayed (A) (Farrow et al., 2008). *P<0.05, **P<0.01, analysis of variance (ANOVA). (B,C) Sections of tumor tissue from mice of each genotype were stained with DHE to assess the level of ROS (Farrow et al., 2008), and with MECA-32 to assess vessel density. For ROS and blood vessel density analysis, a minimum of ten pictures (total magnification, 200ϫ; high-power field) of three tumors from each group was analyzed. *P<0.05, **P<0.01 versus WT, ANOVA. (D-F) Pan02 cells were injected subcutaneously into WT (n=20) and Fbln5RGE/RGE mice (n=21). NAC (7 mg/ml) was added to the drinking water of tumor-bearing WT (n=10) and Fbln5RGE/RGE (n=11) mice for 3 weeks. (D) Tumor volumes were recorded over 3 weeks. ***P<0.001 versus Fbln5RGE/RGE, ANOVA. (E) Sections of tumors from untreated and NAC-treated WT and Fbln5RGE/RGE mice were stained with DHE to determine the level of ROS. Ten pictures (total magnification, 200ϫ) DMM taken of each tumor from WT (n=5), WT+NAC (n=4), Fbln5RGE/RGE (n=5) and Fbln5RGE/RGE+NAC (n=5) animals were used for analysis. (F) Tumor sections were also stained with MECA-32 to analyze vessel density (n=5 for each condition). The number of blood vessels/mm2 is displayed. The results shown are mean±s.e.m. *P<0.01, **P<0.005, ***P<0.001.

was determined by DHE staining. Consistent with the results from treated and untreated Fbln5RGE/RGE mice. The number of MECA- tumors grown in Fbln5–/– animals, tumors from the Fbln5RGE/RGE 32-positive blood vessels in tumors from Fbln5RGE/RGE mice was mice had significantly higher levels of ROS than tumors grown in increased to WT levels in the presence of NAC (Fig. 7F). These WT animals, further confirming the function of Fbln5 in controlling findings further support our hypothesis that the loss of Fbln5 led integrin-induced ROS production (Fig. 7B). We next evaluated to chronic ROS exposure within tumors, causing endothelial cell blood vessel density and observed a significant decrease in death and reduced tumor growth. angiogenesis in tumors grown in Fbln5RGE/RGE mice compared with tumors from their WT counterparts (Fig. 7C). Thus, we show, in DISCUSSION two models (Fbln5–/– and Fbln5RGE/RGE mice), that the loss of Fbln5 A growing tumor interacts with and co-opts the surrounding ECM. function in vivo reduces tumor growth and angiogenesis, and leads To ensure continued growth, tumor cells modify the nature of the to increased ROS production. Results from tumor studies in the ECM to create a microenvironment that is conducive to tumor Disease Models & Mechanisms Fbln5RGE/RGE mice also further validate that integrin binding is progression; this appears to be particularly true for highly crucial for Fbln5-mediated control of ROS generation. desmoplastic tumors such as pancreatic cancer. Matricellular proteins are a crucial component of that process. Until now, the Treatment with an antioxidant rescues angiogenesis and restores effect of the matricellular protein Fbln5 on tumor growth has been tumor growth in Fbln5RGE/RGE mice unclear. Previous reports have shown that fibrosarcoma cells To determine whether increased ROS production in tumors from designed to constitutively express Fbln5 had reduced tumor growth Fbln5RGE/RGE mice was a major antagonist of blood vessel when injected subcutaneously into genetically normal mice (Albig development and tumor growth, we performed tumor implantation et al., 2006). Herein, we report results from the first tumor studies studies in the presence of the antioxidant NAC. Pan02 cells were performed in Fbln5–/– mice. Pancreatic tumor growth was injected subcutaneously into the flank of WT and Fbln5RGE/RGE attenuated in both Fbln5–/– and Fbln5RGE/RGE mice compared with mice, and NAC was added to the drinking water. Tumor volume WT animals. These findings highlight the functional importance was monitored over the course of the experiment. As shown of host-derived Fbln5 and the value of engineered mouse models previously, tumors grew slower in Fbln5RGE/RGE mice than in WT to dissect protein function. We also observed a decrease in mice. However, the tumor growth rate was restored to WT levels angiogenesis in tumors grown in Fbln5–/– mice. This observation in Fbln5RGE/RGE mice treated with NAC (Fig. 7D). Tumors stained was surprising given the recent report that vascular invasion into with DHE demonstrated that treatment with NAC reduced the level implanted polyvinyl sponges was increased in Fbln5–/– mice of ROS in Fbln5RGE/RGE mice (Fig. 7E). compared with WT animals (Albig and Schiemann, 2004; Sullivan To determine whether increased tumor growth in Fbln5RGE/RGE et al., 2007). We found the effect on angiogenesis to be context- mice treated with NAC was due to alleviation of the angiogenic specific such that microvessel density was actually elevated in defect, we analyzed blood vessel density in tumors from NAC- normal non-tumor-bearing pancreas in Fbln5–/– animals compared

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with the pancreas of WT mice. Given that minor increases in ROS (Marikovsky, 2002). Our data support this idea by showing that can stimulate angiogenesis, we speculate that the increased increased ROS production in the tumor microenvironment angiogenesis observed in Fbln5–/– mice under non-tumor effectively reduces tumor growth by diminishing tumor angiogenesis. conditions, where ROS levels are normally low (e.g. normal A remaining question that is under active investigation is: what are pancreas or polyvinyl sponges), might also be a result of increased the cell type(s) that are responsible for ROS production in the absence ROS production (Sullivan et al., 2007). of Fbln5? Given the striking effect on the vascular network in Fbln5–/– Chronic increases in ROS production have been shown to animals, it is likely that endothelial cells participate in integrin- contribute to the onset of certain diseases that are also connected induced ROS generation. However, we also showed that Fbln5 to the loss of Fbln5 expression, such as macular degeneration and functions in fibroblasts to control ROS production. Cancer- cardiovascular disease (Bonomini et al., 2008; Lotery et al., 2006; associated fibroblasts are crucial to tumor progression, and Spencer et al., 2005; Takahashi et al., 2004). Our data would suggest decreasing their level within tumors can slow tumor growth (Pietras that the loss of Fbln5 promotes the progression of these diseases et al., 2008). It is therefore possible that increased production of ROS by causing an increase in ROS production. Also, it is interesting to by fibroblasts within tumors from Fbln5–/– mice resulted in fibroblast note that, although the level of Fbln5 expression is reduced in adult apoptosis and contributed to the decrease in tumor growth. blood vessels compared with neonatal blood vessels, Fbln5 Our results provide further support for the development of drugs expression is induced in adult mice during times of trauma, such that alter the oxidative environment within tumors to control tumor as in response to vascular injury, in atherosclerotic plaques, and in growth. Although much work needs to be carried out to fully interstitial fibroblasts during lung injury repair (Kowal et al., 1999; understand the function of Fbln5 in controlling ROS production, Kuang et al., 2003; Nakamura et al., 1999). These are also instances the validation of its relevance in vivo provides an intriguing target when ROS production is increased; therefore, Fbln5 may be for cancer therapy. required to regulate ROS levels during the remodeling process that occurs after these events. METHODS

DMM It has been reported that Fbln5 inhibits fibronectin-mediated Mice cell spreading, migration and proliferation by competing with Fbln5–/– mice and WT littermates (C57BL/6 ϫ 129/SvEv hybrid fibronectin for b1 integrin binding. Interestingly, binding of Fbln5 background) aged 4-6 months old were used for tumor studies. to b1 integrins does not induce integrin activation, suggesting that The phenotype of the mice was identical to that described Fbln5 functions as a blocking protein to control intracellular previously (Yanagisawa et al., 2002). The generation and phenotypic signaling (Lomas et al., 2007). In support of this idea, it was shown analysis of Fbln5RGE/RGE mice will be described elsewhere. that VSMCs harvested from Fbln5–/– mice exhibit no difference in Investigators interested in obtaining Fbln5–/– or Fbln5RGE/RGE mice proliferation rates compared with WT VSMCs, until treated with should contact Dr Hiromi Yanagisawa. Mice were housed in a platelet-derived growth factor (PDGF). After treatment with PDGF, pathogen-free facility and all experiments were performed Fbln5–/– VSMCs exhibited a twofold increase in proliferation according to a protocol approved by the Institutional Animal Care compared with PDGF-treated WT cells. This effect was curtailed and Use Committee of the University of Texas Southwestern by treatment with recombinant Fbln5 protein, indicating that Fbln5 Medical Center (Dallas, TX). blocks PDGF binding (Spencer et al., 2005). Our data provides further support for this idea by showing that Fbln5 prevents ROS Cell culture production by blocking the interaction between fibronectin and b1 Mouse pancreatic carcinoma cells (Pan02, also known as Panc02), integrins. ROS are prominent signaling molecules that induce the obtained from the Developmental Therapeutics Program, NCI Disease Models & Mechanisms expression of multiple proteins including matrix metalloproteinases (Frederick, MD), murine endothelial cells (bEnd.3), obtained from (MMPs) (Wu, 2006). Recently, it was shown that Fbln5 inhibits Dr Werner Risau, and mouse embryonic fibroblasts (MEFs) MMP-7 expression by an unknown mechanism (Yue et al., 2009). (harvested as described previously) (Yanagisawa et al., 2002) were Given our data, it is possible that Fbln5 inhibits MMP-7 expression all maintained in Dulbecco’s modified Eagle’s medium (DMEM) by limiting ROS production. In addition to this, Fbln5 has also been (Invitrogen, Carlsbad, CA) containing 10% fetal bovine serum (FBS) shown to inhibit VEGF expression, a pro-angiogenic molecule (Gemini Bio-Products, Woodland, CA). All cells were grown in a whose expression can be induced by ROS (Albig and Schiemann, 37°C humidified 5% CO2 atmosphere. MEF passages 1-6 were used 2004; Ushio-Fukai and Alexander, 2004). We speculate that Fbln5 for experiments. controls VEGF expression indirectly by blocking ROS production. Our tumor studies performed in the presence of NAC indicate Tumor growth in vivo that increased ROS levels are the main determinant of decreased Pan02 cells were injected subcutaneously or orthotopically into WT tumor angiogenesis and growth in Fbln5–/– and Fbln5RGE/RGE mice. and Fbln5–/– mice. Cells were harvested from subconfluent cultures Increasing the level of ROS within tumors to control tumor growth is by treatment with 0.25% trypsin and 5 mM EDTA in PBS. After the mechanism of action behind many successful chemotherapeutics trypsinization, the cells were washed once in serum-free medium. and is the basis for a new type of therapy coined ‘oxidation therapy’ Cells were pelleted, washed with PBS, counted, and resuspended (Engel and Evens, 2006; Fang et al., 2007). In addition, recent studies in PBS. Only single-cell suspensions with greater than 90% viability have shown that increasing oxidative stress on the blood vasculature, (as determined by trypan blue exclusion) were used for injections. by specifically inhibiting the breakdown of ROS within endothelial For subcutaneous injections, Pan02 cells (3ϫ106/100 ml) were cells, inhibited tumor growth by decreasing tumor angiogenesis while injected into the flank region of WT and Fbln5–/– mice having no effect on non-tumor, pre-established blood vessel networks (Puolakkainen et al., 2004). After tumor cell injection, the mice were

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monitored for weight, signs of discomfort or morbidity, and tumor of the total number of positive cleaved caspase 3 cells. All samples size. Three to four weeks after tumor cell injection, mice were were visualized with a Nikon Eclipse E600 microscope (Nikon, euthanized. Subcutaneous tumors were excised, weighed, and the Lewisville, TX). All fluorescent images were captured with a tumor samples were frozen in liquid nitrogen or fixed in methyl Photometric Coolsnap HQ camera and analyzed with either Carnoy’s fixative for histological analysis. N-acetyl cysteine (NAC) Metamorph software (Universal Imaging Corporation, (7 mg/ml) was given in the drinking water for the length of the Downington, PA) or the Nikon Imaging software Elements. experiment. Orthotopic tumor cell injection was carried out as described ROS detection previously (Arnold et al., 2008). Briefly, the mice were anesthetized Frozen tumors were sectioned (10 mm) and treated with with isoflurane, which was maintained throughout the time of dihydroethidium (DHE; Molecular Probes, Eugene, OR). 50 ml of surgery. A small left abdominal incision was made and the spleen 5 mM DHE was added to each tumor section, coverslipped, and was exposed to access the pancreas. Tumor cells (5ϫ105/50 ml) in incubated at 37°C in a humidified incubator for 30 minutes. PBS were injected into the tail of the pancreas. A successful Nuclear Red fluorescence was visualized and images captured using subcapsular intrapancreatic injection of tumor cells was identified the Photometric Coolsnap HQ camera. Fluorescence intensity was by the appearance of a fluid bleb without intraperitoneal leakage. quantified using Metamorph software, as described above. To The animals tolerated the surgical procedure well, and no visualize ROS in MEFs, cells were grown on slides that were pre- anesthesia-related deaths occurred. Four weeks after injection, mice coated with fibronectin (F1141, Sigma-Aldrich) or collagen type 1 were euthanized and the peritoneal cavity (including the liver and (354236, BD Biosciences) at 5 mg/ml. After 24 hours the media was spleen), inguinal and axillary regions, kidneys, thoracic cavity removed; cells were washed once with warm PBS and then (including the lungs and heart), and brain were screened for incubated with 10 mM of 2Ј-7Ј-dicholordihydrofluorescein diacetate metastases by visual inspection under a dissecting microscope and (DCF-DA) (D399, Molecular Probes) in warm PBS with 5.5 mM then confirmed by histology. The entire pancreas containing the glucose for 10 minutes at 37°C. The DCF-DA was removed and

DMM tumor lesion was harvested and weighed. replaced with DMEM with 10% FCS for an additional 10 minutes at 37°C. Cells were again washed with warm PBS, fixed with 10% Immunohistological analysis formalin for 5 minutes, and mounted with ProLong Gold antifade Immunohistochemistry was performed on frozen or fixed tissue reagent with DAPI (Invitrogen). Images and fluorescence were sections. For immunohistochemistry on fixed tissues, tissues were quantified and normalized to the cell number, as described above. deparaffinized in xylene, followed by a graded series of ethanol ROS levels were also detected in cells by use of a fluorometer. MEFs exchanges, and rehydrated in PBS containing 0.2% Tween (PBST). or bEnd.3 cells (2ϫ104) were plated on 96-well plates that were The primary antibodies used were: rabbit anti-phosphorylated pre-coated with either fibronectin or collagen (5 mg/ml). Cells were histone H3 (phospho-H3) (Upstate Biotechnology, Inc., Lake Placid, allowed to adhere for 1 hour and then media was removed and NY); rat anti-mouse endothelial cell marker, MECA-32 (Hallmann DCF-DA (20 mM) added for 1 hour at 37°C. DCF-DA was removed et al., 1995) (Developmental Studies Hybridoma Bank, University and the cells were washed once with PBS. Fluorescence was of Iowa, Iowa City, IA); rabbit anti-mouse smooth muscle actin measured with an excitation wavelength of 480 nm and emission (SMA) (RB-9010-P, LabVision, Fremont, CA); goat anti-8- wavelength of 520 nm. Cells were treated with either full-length hydroxydeoxyguanosine (8-OHdG) (AB5830, Chemicon, Billerica, recombinant Fbln5, NH2-terminal truncation mutant Fbln5 (a MA); rabbit anti-gH2AX (NB100-2280, Novus Biologicals, Littleton, generous gift from Elaine C. Davis, McGill University) or anti-b1 CO); and rabbit anti-mouse Fbln5 (BSYN1923) (Zheng et al., 2007). integrin blocking antibody (CD29 #555002, BD Biosciences), at 10 Disease Models & Mechanisms All antibodies were used at a concentration of 5-10 mg/ml. mg/ml, for 15 minutes at 37°C before plating in wells. To block the Fluorescent sections were mounted with ProLong Gold antifade ROS signal, N-acetyl cysteine (NAC) (10 mM) was added to the reagent with DAPI (Invitrogen). Primary antibody binding was DCF-DA reagent prior to adding to cells. Each experiment was detected with peroxidase-conjugated or fluorophore-conjugated performed in triplicate. (fluorescein FITC or Cy3) secondary antibodies (Jackson Immunoresearch, West Grove, PA). siRNA knockdown of Fbln5 TUNEL staining was performed using a DeadEnd Fluorometric bEnd.3 endothelial cells were plated in six-well plates and allowed TUNEL System (Promega, Madison, WI), according to the to adhere overnight. Cells were transfected with either pre-designed manufacturer’s protocol. siRNAs directed against Fbln5 or a scrambled sequence (Sigma- Aldrich) using the N-TER Nanoparticle siRNA Transfection System Quantification of immunohistochemistry (Sigma-Aldrich). Twenty-four hours after transfection, cells were Multiple fluorescent images per sample were captured under trypsinized and a portion was plated in 96-well plates for ROS identical conditions (i.e. room temperature, exposure). The detection. The remaining portion of cells was resuspended in percentage of pixels exceeding the threshold value (background) sample buffer (Bio-Rad) for western blot analysis. Western blots was calculated automatically by the software (% average intensity). were performed using standard protocols. Mean blood vessel counts and area were measured either by blind hand counting or by using Metamorph’s ‘Integrated Morphometry Statistical analysis Analysis’ tool. For analysis of endothelial cell death, tumor sections Statistical analyses between genotypes and various conditions were stained for cleaved caspase 3 and co-localized with MECA- were performed using Student’s t-test or ANOVA. P values less 32. Double positive cells were counted and taken as a percentage than 0.05 were considered statistically significant.

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Disease Models & Mechanisms protein. Nat. Genet. 36, 178-182. suitable target for anti-tumor therapy in pancreatic, and other, cancers. Lomas, A. C., Mellody, K. T., Freeman, L. J., Bax, D. V., Shuttleworth, C. A. and doi:10.1242/dmm.005322 Kielty, C. M. (2007). Fibulin-5 binds human smooth-muscle cells through alpha5beta1 and alpha4beta1 integrins, but does not support receptor activation. Biochem. J. 405, 417-428. ACKNOWLEDGEMENTS Lotery, A. J., Baas, D., Ridley, C., Jones, R. P., Klaver, C. C., Stone, E., Nakamura, T., This work was supported in part by grants from the NIH (R01 HL07115 to H.Y., R01 Luff, A., Griffiths, H., Wang, T. et al. (2006). Reduced secretion of fibulin 5 in age- HL070187 to T.F. and R01 CA118240 to R.A.B.), The Welch Foundation (to H.Y.), the related macular degeneration and cutis laxa. Hum. Mutat. 27, 568-574. Effie Marie Cain Scholarship in Angiogenesis Research (to R.A.B.) and The Mantovani, A., Allavena, P., Sica, A. and Balkwill, F. (2008). Cancer-related Department of Surgery, UT-Southwestern Medical Center (to R.A.B.). We gratefully inflammation. 454, 436-444. acknowledge the support and critical evaluation of this work by members of the Nature Marikovsky, M. (2002). Thiram inhibits angiogenesis and slows the development of Brekken and Yanagisawa laboratories. Deposited in PMC for release after 12 months. experimental tumours in mice. Br. J. Cancer 86, 779-787. COMPETING INTERESTS Nakamura, T., Ruiz-Lozano, P., Lindner, V., Yabe, D., Taniwaki, M., Furukawa, Y., The authors declare no competing financial interests. Kobuke, K., Tashiro, K., Lu, Z., Andon, N. L. et al. (1999). DANCE, a novel secreted RGD protein expressed in developing, atherosclerotic, and balloon-injured arteries. J. AUTHOR CONTRIBUTIONS Biol. Chem. 274, 22476-22483. M.K.S. planned and performed experiments, analyzed data and wrote the paper; Nguyen, A. D., Itoh, S., Jeney, V., Yanagisawa, H., Fujimoto, M., Ushio-Fukai, M. S.L.C. maintained the mouse colony and performed experiments; G.K. performed and Fukai, T. (2004). Fibulin-5 is a novel binding protein for extracellular superoxide experiments and analyzed data; K.O. and T.F. performed experiments and analyzed dismutase. Circ. Res. 95, 1067-1074. data; H.Y. planned the project and analyzed data; R.A.B. planned the project, Pietras, K., Pahler, J., Bergers, G. and Hanahan, D. (2008). Functions of paracrine analyzed data and wrote the paper. PDGF signaling in the proangiogenic tumor stroma revealed by pharmacological SUPPLEMENTARY MATERIAL targeting. PLoS Med. 5, e19. Supplementary material for this article is available at Pozzi, A., Moberg, P. E., Miles, L. A., Wagner, S., Soloway, P. and Gardner, H. A. http://dmm.biologists.org/lookup/suppl/doi:10.1242/dmm.003707/-/DC1 (2000). Elevated matrix metalloprotease and angiostatin levels in integrin alpha 1 knockout mice cause reduced tumor vascularization. Proc. Natl. Acad. Sci. USA 97, Received 22 May 2009; Accepted 21 August 2009. 2202-2207.

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