Skip to main content
PMC home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2012 Oct 1.
Published in final edited form as: Mol Cancer Res. 2011 Aug 19;9(10):1294–1304. doi: 10.1158/1541-7786.MCR-11-0023

MT1-MMP cooperates with KrasG12D to promote pancreatic fibrosis through increased TGF-β signaling

Seth B Krantz 1, Mario A Shields 2, Surabhi Dangi-Garimella 2, Eric C Cheon 1, Morgan R Barron 1, Rosa F Hwang 6, M Sambasiva Rao 3, Paul J Grippo 1,4, David J Bentrem 1,4,5, Hidayatullah G Munshi 2,4,5,*
PMCID: PMC3196812  NIHMSID: NIHMS320257  PMID: 21856775

Abstract

Pancreatic cancer is associated with a pronounced fibrotic reaction that was recently shown to limit delivery of chemotherapy. To identify potential therapeutic targets to overcome this fibrosis, we examined the interplay between fibrosis and the key proteinase membrane type 1-matrix metalloproteinase (MT1-MMP, MMP-14), which is required for growth and invasion in the collagen-rich microenvironment. In this report we show that compared to control mice (Kras+/MT1-MMP-) that express an activating KrasG12D mutation necessary for pancreatic cancer development, littermate mice that express both MT1-MMP and KrasG12D (Kras+/MT1-MMP+) developed a greater number of large, dysplastic mucin-containing papillary lesions. These lesions were associated with a significant amount of surrounding fibrosis, increased α-smooth muscle actin(+) cells in the stroma, indicative of activated myofibroblasts, and increased Smad2 phosphorylation. To further understand how MT1-MMP promotes fibrosis, we established an in vitro model to examine the effect of expressing MT1-MMP in pancreatic ductal adenocarcinoma (PDAC) cells on stellate cell collagen deposition. Conditioned media from MT1-MMP-expressing PDAC cells grown in 3D collagen enhanced Smad2 nuclear translocation, promoted Smad2 phosphorylation and increased collagen production by stellate cells. Inhibiting the activity or expression of the TGF-β type I receptor in stellate cells attenuated MT1-MMP conditioned media-induced collagen expression by stellate cells. Additionally, a function-blocking anti-TGF-β antibody also inhibited MT1-MMP conditioned media-induced collagen expression in stellate cells. Overall, we demonstrate that the bona fide collagenase MT1-MMP paradoxically contributes to fibrosis by increasing TGF-β signaling and that targeting MT1-MMP may thus help to mitigate fibrosis.

Keywords: MT1-MMP, Fibrosis, Pancreatic Cancer, Stellate Cells, TGF-β

INTRODUCTION

Pancreatic ductal adenocarcinoma (PDAC), the fourth leading cause of cancer-related death in the US, is associated with an intense fibrotic reaction (1-4). This encasing tissue, recently shown to limit the efficacy of current chemotherapy (5), is composed of interstitial extracellular matrix (ECM), stellate cells, and other stromal cells that can outnumber local tumor cells (6, 7). The stellate cells are usually differentiated into an activated myofibroblastic phenotype and exhibit increased expression of α-smooth muscle actin (6-8). They are the primary source of type I collagen that is present in the fibrotic reaction and can promote cancer cell proliferation and invasion in both in vitro and in vivo models (9, 10). PDAC tumors, particularly in areas of fibrosis, demonstrate increased TGF-β1 signaling (11, 12), which is also involved in stellate cell activation (13). Given the strong correlation between TGF-β1 and type I collagen in human pancreatic specimens (14), TGF-β1 may be directly responsible for the fibrotic reaction in PDAC tumors by mediating collagen production by stellate cells.

Membrane type 1-matrix metalloproteinase (MT1-MMP, MMP-14), a primary interstitial collagenase, is associated with invasion and metastases in several human cancers (15, 16). Gene and protein expression studies of stromal and neoplastic cells of primary PDAC tumors have shown that MT1-MMP is overexpressed in pancreatic cancer cells (11, 17, 18), and that expression of MT1-MMP is enhanced in metastatic PDAC lesions compared to the primary tumors (19). Animal studies support MT1-MMP as a primary regulator of interstitial collagenolysis, as mice deficient in MT1-MMP have severe growth defects due to the inability to process interstitial collagens (20, 21). As the primary regulator of interstitial collagenolysis, MT1-MMP is essential for growth within the collagen-dense in vivo microenvironment (22). Moreover, MT1-MMP overexpression in the mouse mammary gland using a transgenic model was by itself sufficient to induce adenocarcinoma (23). Interestingly, though a collagenase, MT1-MMP has been associated with increased fibrosis in breast and squamous cell cancers and in non-malignant MDCK cells (23-25) and therefore may also contribute to fibrosis within pancreatic cancer, though this remains to be determined. Despite the importance of MT1-MMP in cancer progression and its association with both primary and metastatic PDAC, the contribution of MT1-MMP to pancreatic cancer development remains to be defined.

In this report we examined the role of MT1-MMP in pancreatic tumor development by generating transgenic mice with targeted expression of human MT1-MMP in elastase (EL) positive cells in the pancreas, which were subsequently crossed with EL-KrasG12D mice. Compared to littermate EL-KrasG12D/MT1-MMP- control mice, mice expressing both MT1-MMP and KrasG12D developed large, dysplastic, mucin-containing papillary lesions with pronounced surrounding fibrosis, increased TGF-β signaling and increased activation of stellate cells. To define a potential mechanism to explain these findings, we established an in vitro model to examine the effect of overexpressing MT1-MMP in PDAC cells on stellate cell collagen deposition. We demonstrate that the conditioned media from MT1-MMP-expressing PDAC cells increased TGF-β signaling in stellate cells to drive stellate cell collagen production. Overall, we demonstrate that the bona fide collagenase MT1-MMP potentiates TGF-β signaling within the tumor microenvironment and contribute to fibrosis in vivo.

MATERIALS AND METHODS

Chemicals/Reagents

MT1-MMP antibody was purchased from Abcam (Cambridge, MA), Smad2 and p-Smad2(Ser465/467) antibodies from Cell Signaling (Danvers, MA), type I collagen antibody from Southern Biotech (Birmingham, AL), and α-tubulin and TGF-β receptor type I (TβRI) antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). Secondary antibodies were purchased from Sigma (St. Louis, MO). Type I collagen was purchased from BD Biosciences (Franklin Lakes, NJ), and TβRI inhibitor SB431542 from Tocris (Ellisville, MO). Nucleofector electroporation kit was purchased from Lonza (Walkersville, MD).

Transgenic Mice

All animal work was conducted in compliance with the Northwestern University IACUC guidelines. TRE-MT1-MMP transgenic mice, in which MT1-MMP expression is under the control of seven tet-responsive elements (TREs) upstream of a minimal CMV promoter (26, 27), were created by the Transgenic Core Facility at Northwestern University. The TRE-MT1-MMP mice were crossed with EL-tTA mice, kindly provided by Dr. Eric Sandgren (26), to generate EL-tTA/TRE-MT1-MMP bigenic mice. In EL-tTa mice, the transactivator tTa is expressed downstream of elastase (EL) promoter, thus enabling targeting of MT1-MMP to pancreatic acinar and centroacinar cells. The bigenic mice were further crossed with EL-KrasG12D mice, which express constitutively active mutant Kras in the pancreatic acinar cells (26, 27). The background of the majority of the mice was B6/B6/B6, with a small number of the mice being B6/FVB/B6 or B6/FVB/FVB. The trigenic mice were raised in the same cage as their littermates, and all comparisons were made between littermates with the same genetic background. Kras+/MT1-MMP- mice were EL-Kras+/EL-tTA+/TRE-MT1-MMP-, EL-Kras+/EL-tTA-/TRE-MT1-MMP+, or EL-Kras+/EL-tTA-/TRE-MT1-MMP-, while Kras+/MT1-MMP+ mice were EL-Kras+/EL-tTA+/TRE-MT1-MMP+. Study mice were euthanized at 9-11 months of age. No mice developed weight loss, suggesting that there was no pronounced pancreatic insufficiency, or died from disease.

Histologic analysis

Samples were evaluated by M.S. Rao, a board-certified pathologist who was blinded to the genotype of the mice. Using a 1 cm × 1 cm reticle, lesion count was determined by counting all cystic lesions greater than 100 μm in maximum diameter, while lesion size was determined by measuring the maximum diameter of individual lesions. Lesion volume was determined utilizing the measured diameter and assuming a spherical geometry for each lesion, with the volume being equal to (4/3)πr3. The samples were trichrome stained and whole gland fibrosis was scored by a pathologist associated with our Pathology Core Facility. This pathologist was blinded to the mouse genotype, and the mice were scored according to the percentage of the entire gland containing pathologic fibrosis. The extent of acinar to ductal metaplasia (ADM), defined by vacuolization of the normal acini with formation of abnormal ducts along with evidence of dysplasia and fibrosis, was assessed at 40x magnification and a score of less than 25%, 25%-75%, or greater than 75% of the pancreas containing ADM was assigned to each mouse. The scoring was performed by S.B. Krantz who was blinded to mouse genotype, and was independently confirmed by P.J. Grippo, who has extensive experience in these transgenic mouse models. Paired littermate control (Kras+/MT1-MMP-) and positive (Kras+/MT1-MMP+) mice were alcian blue stained by the pathology core facility to assess the presence of mucin within the lesions. All histologic comparisons were made between littermates raised in the same cage, all with the same genetic background.

Immunohistochemistry

Pancreatic tissue specimens from paired littermate Kras+/MT1-MMP- and Kras+/MT1-MMP+ were stained for p-Smad2, α-SMA (Abcam), TUNEL (ApopTag kit S7100; Chemicon), proliferating cell nuclear antigen (PCNA; Santa Cruz), MT1-MMP, and CK-19 (University of Iowa). Antigen retrieval for p-Smad2, α-SMA, PCNA, MT1-MMP, and CK-19 was performed as previously described (28). The pSmad2 3101 antibody was validated for immunohistochemisty by paraffin embedding pancreatic cancer cells treated with TGF-β1 +/- SB431542 and processing for pSmad2 as for the mouse tissue samples. TUNEL staining was performed according to the kit instructions. Photographs for quantitative comparison were taken using a Carl Zeiss Axiovert 200 microscope and camera. The paired images were analyzed simultaneously using Image J software to determine the percentage of cells with nuclear staining for p-Smad2, TUNEL, and PCNA.

Cell culture

AsPC1 and Panc1 cells were obtained from American Type Culture Collection (Manassas, VA), while human stellate cells have been described previously (9). Panc1 and AsPC1 cells were tested in December 2009 and June 2010 by STR profiling at the Johns Hopkins Genetic Resources Core Facility. Stellate cells were verified by R.F. Hwang prior to shipment in January 2010.

Conditioned Media

AsPC1 and Panc1 cells expressing MT1-MMP were generated as detailed previously (24). Cells expressing control vector or MT1-MMP were grown in 3D type I collagen gels (2.2 mg/ml), and allowed to condition the media for 96 hours to generate Vector-and MT1-MMP-conditioned media.

Immunoblotting & Immunofluorescence

Immunoblotting for type I collagen, p-Smad2, MT1-MMP, TβRI and α-tubulin was done as previously described (11). The effect on Smad2 nuclear translocation following treatment of stellate cells with Vector- and MT1-MMP-conditioned media was done as described previously (29).

Stellate cell collagen expression

Vector- and MT1-MMP-conditioned media were added for 72 hours to stellate cells grown on tissue culture plastic, which were then processed for either RNA or total protein as previously published (11). Relative collagen production in stellate cells treated with MT1-MMP-conditioned media was normalized to levels present in stellate cells treated with Vector-conditioned media using the Comparative CT method and GAPDH as normalization control (11).

For TβRI inhibition experiments with siRNA, stellate cells were transfected with control siRNA or siRNA against TβRI (Ambion) using a nucleofector kit. The cells were allowed to recover for 24 hours and were then grown in conditioned media for an additional 48 hours before being assayed for collagen mRNA expression. The TβRI kinase activity in the stellate cells was also blocked using SB431542. In addition, the Vector- and MT1-MMP-conditioned media were incubated with function blocking anti-TGF-β1-3 antibody (R&D Systems) or isotype-matched IgG control antibody prior to adding the media to the stellate cells to examine the effect on collagen production.

Statistical Analysis

Comparisons of in vivo data were made using paired Wilcoxon signed rank tests, with the pairs representing littermate Kras+/MT1-MMP- and Kras+/MT1-MMP+ mice raised in the same cage. For categorical analyses, Chi-square analysis was used. In vitro results were compared using paired t-test analysis. Error bars represent standard error of the mean. All analyses were performed on GraphPad Prism 5 for Mac OS X.

RESULTS

MT1-MMP expression in Kras G12D mice increases acinar to ductal metaplasia (ADM)

We had previously shown that human pancreatic tumors demonstrate increased MT1-MMP expression in areas of fibrosis (11). To understand the contribution of MT1-MMP to pancreatic cancer development, we created TRE-MT1-MMP transgenic mice in which human MT1-MMP expression is under the control of seven tet-responsive elements upstream of a minimal CMV promoter (26, 27). The TRE-MT1-MMP mice were crossed with EL-tTA mice, which allows targeting of MT1-MMP to the acinar and centroacinar cells in the pancreas (26), to generate EL-tTA+/TRE-MT1-MMP+ bigenic mice. Even though human MT1-MMP expression was successfully induced in the pancreas (Supplemental Fig. S1), MT1-MMP alone was not sufficient to cause precancerous or cancerous changes. These mice had histologically normal pancreas. Since published reports have established the requirement of mutant KrasG12D for pancreatic cancer development (30-33), we generated mice that express both activated KrasG12D and MT1-MMP in the pancreas. The bigenic EL-tTA+/TRE-MT1-MMP+ were crossed with EL-KrasG12D mice to create EL-KrasG12D+/EL-tTA+/TRE-MT1-MMP+ (Kras+/MT1-MMP+) and Kras+/MT1-MMP- mice, which were EL-KrasG12D+/EL-tTA+/TRE-MT1-MMP-, EL-KrasG12D+/EL-tTA-/TRE-MT1-MMP+ or EL-KrasG12D+/EL-tTA-/TRE-MT1-MMP- mice (Fig. 1A). Note that only mice that were positively genotyped for both EL-tTA and MT1-MMP expressed human MT1-MMP in the mouse pancreas. Compared to littermate Kras+/MT1-MMP- control mice, the Kras+/MT-MMP+ mice demonstrated increased MT1-MMP protein expression (Fig. 1B) and developed significantly increased ADM, as demonstrated by loss of eosinophilic granules, vacuolization of the acini, development of ductular structures, and increase in the surrounding stroma (Figs. 1C and 1D).

Figure 1. MT1-MMP expression in KrasG12D mice increases acinar to ductal metaplasia (ADM).

Figure 1

Open in a new tab

A. The TRE-MT1-MMP (TRE-MT) mice were generated as detailed in the Materials and Methods and crossed with EL-tTA mice. The EL-tTA+/TRE-MT1-MMP+ mice were crossed with EL-KrasG12D mice to generate mice expressing both MT1-MMP and KrasG12D in the pancreas (EL-Kras+/EL-tTA+/TRE-MT+ = Kras+/MT1-MMP+) or littermate control mice that expressed only KrasG12D and not MT1-MMP (EL-Kras+/EL-tTA+/TRE-MT-, EL-Kras+/EL-tTA-/TRE-MT+, or EL-Kras+/EL-tTA-/TRE-MT- = Kras+/MT1-MMP-). The mRNA samples from pancreas of control Kras+/MT1-MMP- mice and Kras+/MT1-MMP+ mice were analyzed for human (h) MT1-MMP, and mouse (m) GAPDH by RT-PCR. B,C. Shown here are representative comparisons of pancreas from littermates at 9-11 months of age demonstrating MT1-MMP expression by immunohistochemistry and the degree of ADM as seen by H&E. D. The extent of ADM was quantified in these mice as described in the Materials and Methods. Shown here are the number of Kras+/MT1-MMP- and Kras+/MT1-MMP+ mice with less than 25%, 25%-75%, or greater than 75% of their pancreas containing ADM (n=34 Kras+/MT1-MMP- mice, 32 Kras+/MT1-MMP+ mice, p<0.005).

MT1-MMP expression in Kras G12D mice increases the number and size of precancerous lesions in the pancreas with a corresponding increase in proliferation

As expression of KrasG12D in the pancreas was previously shown to cause mucinous cystic neoplasms (26), we examined the effect of MT1-MMP expression on the nature, size and number of pancreatic lesions. The lesions in Kras+/MT1-MMP+ had papillary features with a high degree of cellular atypia and contained mucin (Supplemental Fig. S2A), features very similar to those seen in human intraductal papillary mucinous neoplasms (IPMNs) (34). Moreover, compared to Kras+/MT1-MMP- control mice, the Kras+/MT1-MMP+ mice developed significantly more (Fig. 2A) and larger lesions (Fig. 2B). Kras+/MT1-MMP+ mice had a median of 3 lesions greater than 100 μm in maximum diameter, compared with a median of 0.4 lesions greater than 100 μm in maximum diameter in the control Kras+/MT1-MMP- mice (Fig. 2A, right). The median size of lesions in the Kras+/MT1-MMP+ was 340 μm, compared with a median size of 75 μm in littermate control Kras+/MT1-MMP- mice (Fig. 2B, right). Assuming a spherical shape for each lesion, we calculated lesion volume and found that the Kras+/MT1-MMP+ mice had lesions that were on average nearly 20-fold larger than lesions in the Kras+/MT1-MMP- mice (p<0.05). To account for extremely large lesions or a very high number of lesions in a single mouse creating a confounding outlier effect we also grouped the mice as having small lesions, medium-sized lesions, or large lesions (<750 μm, 750-2500 μm, >2500 μm); or as having no lesions, one to three lesions or greater than three lesions. Significantly more Kras+/MT1-MMP+ mice had both increased lesion counts along with more medium- and large-sized lesions than did the Kras+/MT1-MMP- mice (p<0.005). We also examined the effect of MT1-MMP expression on proliferation and apoptosis. Compared to littermate control Kras+/MT1-MMP- mice, Kras+/MT1-MMP+ mice showed a statistically significant increase in the number of actively proliferating cells as determined by PCNA staining (Figs. 2C). In contrast, there was minimal TUNEL staining in both Kras+/MT1-MMP+ and Kras+/MT1-MMP- mice, which was not statistically different between the two groups of mice (Supplemental Fig. S2B). Also, while no lesion showed areas of frank invasion, there were several areas that, when stained for the pancreatic epithelial marker cytokeratin-19, demonstrated individual cells within the surrounding stroma of Kras+/MT1-MMP+ mice (Supplemental Fig. S3A).

Figure 2. MT1-MMP expression in KrasG12D mice increases the number and size of precancerous lesions in the pancreas with a corresponding increase in proliferation.

Figure 2

Open in a new tab

Kras+/MT1-MMP- (EL-Kras+/EL-tTA+/TRE-MT-, EL-Kras+/EL-tTA-/TRE-MT+, or EL-Kras+/EL-tTA-/TRE-MT-) and Kras+/MT1-MMP+ (EL-Kras+/EL-tTA+/TRE-MT+) mice were generated as detailed in the materials and methods. A. Representative H&E comparison of pancreas from 2 pairs of littermates at 9-11 months of age showing the effect of MT1-MMP on lesion number. Total number of lesions greater than 100 μm in paired Kras+/MT1-MMP- and Kras+/MT1-MMP+ littermate (n=14 pairs, p< 0.005) was determined by microscopic examination as detailed in the Materials and Methods. B. Representative H&E comparison of pancreas from 2 pairs of littermates at 9-11 months of age showing the effect of MT1-MMP on lesion size. Average lesion size in paired Kras+/MT1-MMP- and Kras+/MT1-MMP+ littermate (n=14 pairs, p< 0.02) was determined by microscopic examination as detailed in the Materials and Methods. C. Effect of MT1-MMP on the number of proliferating cells (brown staining) as determined by immunohistochemistry for PCNA and the percentage of PCNA positive cells in paired Kras+/MT1-MMP- and Kras+/MT1-MMP+ littermates was quantified (n=10 pairs, p<0.005).

MT1-MMP expression in KrasG12D mice promotes pancreatic fibrosis, enhances Smad2 phosphorylation and increases the number of α-SMA+ cells

Interestingly, we also found that compared to control Kras+/MT1-MMP- mice, Kras+/MT1-MMP+ littermate mice developed significantly increased fibrosis surrounding their lesions (Figs. 3A and 3B). Trichrome (blue) staining showed that there was increased collagen deposition throughout the gland in the Kras+/MT1-MMP+ mice compared to littermate control Kras+/MT1-MMP- mice (Fig. 3C) and significantly more Kras+/MT1-MMP+ mice had pathologic fibrosis in a moderate to high percent of their pancreas compared to their littermate controls (Fig. 3D). Since there is a strong correlation between TGF-β levels and type I collagen expression in human pancreatic specimens (11, 12, 14), we analyzed the tumors for evidence of TGF-β signaling by staining for p-Smad2. As shown in Figs. 4A and 4B, lesions from Kras+/MT1-MMP+ mice demonstrate increased p-Smad2 staining compared to littermate control Kras+/MT1-MMP- mice. This increase in p-Smad2 was not limited to the tumors, but was also seen in the stromal compartment (Supplemental Fig. S3B). As pancreatic stellate cells have been implicated as the main contributors to fibrosis in vivo (8-10), with the activated stellate cells staining positive for α-smooth muscle actin (α-SMA), we stained the pancreatic specimens from Kras+/MT1-MMP-and Kras+/MT1-MMP+ mice for α-SMA expression. As shown in Fig. 4C, Kras+/MT1-MMP+ mice demonstrate increased α-SMA staining in the stroma compared to the control Kras+/MT1-MMP- mice.

Figure 3. MT1-MMP expression in KrasG12D mice promotes pancreatic fibrosis.

Figure 3

Open in a new tab

A, C. Shown here is representative H&E (A) and trichrome (C, collagen is blue staining) comparisons of pancreatic tissue samples from paired Kras+/MT1-MMP- (EL-Kras+/EL-tTA+/TRE-MT-, EL Kras+/EL-tTA-/TRE-MT+, or EL-Kras+/EL-tTA-/TRE-MT-) and Kras+/MT1-MMP+ (EL-Kras+/EL-tTA+/TRE-MT+) littermates at 9-11 months of age. B. The average amount of associated fibrosis surrounding each lesion (n=14 pairs, p< 0.005) in paired Kras+/MT1-MMP- and Kras+/MT1-MMP+ littermates. D. The number of Kras+/MT1-MMP- and Kras+/MT1-MMP+ mice with a low, moderate, or high amount of their pancreas containing pathologic fibrosis (n=34 Kras+/MT1-MMP- mice, 32 Kras+/MT1-MMP+ mice, p<0.0001).

Figure 4. MT1-MMP expression in KrasG12D mice enhances Smad2 phosphorylation and increases α-SMA(+) cells.

Figure 4

Open in a new tab

A, B. Representative IHC staining at low and high magnification for p-Smad2 (nuclear brown staining) and graph of the percentage of p-Smad2 positive staining cells in paired Kras+/MT1-MMP- and Kras+/MT1-MMP+ littermates at 9-11 months of age (n=10 pairs, p< 0.005). C. IHC staining of α-SMA (brown staining) of pancreas from paired Kras+/MT1-MMP- (EL-Kras+/EL-tTA+/TRE-MT-, EL-Kras+/EL-tTA-/TRE-MT+, or EL-Kras+/EL-tTA-/TRE-MT-) and Kras+/MT1-MMP+ (EL-Kras+/EL-tTA+/TRE-MT+) littermates at 9-11 months of age.

MT1-MMP expression in pancreatic cancer promotes collagen production by pancreatic stellate cells in an in vitro model system

Since our in vivo findings show increased pancreatic fibrosis associated with increased Smad2 phosphorylation and an increase in α-SMA(+) cells, we hypothesized that MT1-MMP increases TGF-β signaling to cause stellate cells to produce more collagen. To test this hypothesis, we established an in vitro model to examine the effect of expressing MT1-MMP in pancreatic cancer cells on stellate cell collagen deposition (Fig. 5A). We successfully generated human PDAC cells expressing an empty vector or MT1-MMP (Fig. 5B), grew them in 3D type I collagen gels, and allowed them to condition the media for 96 hours. The conditioned media was then added for 72 hours to stellate cells to examine the effect on collagen mRNA production. Relative to treatment with conditioned media from vector expressing control PDAC cells, treatment with MT1-MMP-conditioned media increased stellate cell type I collagen mRNA (Fig. 5C) and protein expression (Fig. 5D).

Figure 5. MT1-MMP expression in pancreatic cancer promotes collagen production by pancreatic stellate cells in an in vitro model system.

Figure 5

Open in a new tab

A. Schematic representation of in vitro culture model utilizing PDAC cells grown in 3D collagen gels (2.2 mg/ml) and allowed to condition the media for 96 hours to generate Vector- and MT1-MMP-conditioned media, which was then used to treat human pancreatic stellate cells. B. Western blot of PDAC (AsPC1 and Panc1) cells expressing control vector or MT1-MMP. C, D. The PDAC conditioned media were added to human stellate cells grown on tissue culture plastic for 72 hours to examine the effect on collagen production by real time PCR (*, p<0.05) and western blot analysis. The results are representative of at least 3 independent experiments.

MT1-MMP expression in pancreatic cancer cells increases TGF-β signaling in pancreatic stellate cells to promote collagen production by stellate cells

We next examined whether TGF-β signaling mediated the effect of conditioned media from MT1-MMP expressing cells on stellate cell collagen production. Initially, we examined the effect of conditioned media on Smad signaling by determining the effect on nuclear translocation of Smad2 in stellate cells. Similar to effect of exogenous TGF-β treatment of stellate cells (Supplemental Fig. S4A), conditioned media from MT1-MMP-expressing AsPC1 and Panc1 cells enhanced Smad2 nuclear translocation at 30 minutes (Fig. 6A, top). We also examined the effect on Smad2 phosphorylation using a p-Smad2(Ser465/467) antibody. As shown in Fig. 6A (bottom), there was increased Smad2 phosphorylation at 30 minutes following treatment with MT1-MMP-conditioned media, indicating that the media conditioned by MT1-MMP-expressing cancer cells contains active TGF-β.

Figure 6. MT1-MMP expression in pancreatic cancer increases TGF-β signaling in pancreatic stellate cells to promote collagen production by stellate cells.

Figure 6

Open in a new tab

A, top. Stellate cells were plated onto glass coverslips, serum starved, treated with Vector- or MT1-MMP-conditioned media for 30 minutes and processed for immunofluorescence using anti-Smad2 and Alexa-Fluor-488-labeled antibodies. The nuclei were co-stained with DAPI and the immunofluorescence signal detected using a Zeiss microscope. A, bottom. Stellate cells were treated with Vector- or MT1-MMP-conditioned media for 30 minutes. The cell lysates were then probed for p-Smad2(Ser465/467), total Smad2, and α-tubulin as loading control. B. Stellate cells were transfected with control siRNA (siCtl) or with TGF-β type I receptor siRNA (siTβRI), allowed to recover for 48 hours and then treated with PDAC conditioned media for an additional 48 hours. Lysates were analyzed for TβRI and α-tubulin expression by western blotting (top). The effect on collagen mRNA was quantified with real time PCR and normalized to the levels present in siCtl-transfected stellate cells treated with Vector-conditioned media (*, p<0.05, **p<0.01, ns, not significant) (bottom). C. Stellate were cells grown in PDAC-conditioned media, treated with either DMSO or TβRI inhibitor SB431542 (10 μM) for 48 hours, and the effect on collagen mRNA was then determined with real time PCR and normalized to the levels present in DMSO-treated stellate cells grown in Vector-conditioned media (*, p<0.05, **p<0.01, ns, not significant). D. PDAC-conditioned media were incubated with isotype control IgG or anti-TGF-β1-3 function blocking antibody (2 μg/mL) for 30 min prior to adding to stellate cells. The effect on collagen mRNA at 48 hours was then determined with real time PCR and normalized to the levels present in stellate cells treated with Vector-conditioned media containing control IgG antibody (*, p<0.05, **p<0.01, ns, not significant). The results are representative of at least 3 independent experiments.

We next assessed whether attenuating TGF-β signaling in stellate cells blocked MT1-MMP-conditioned media-induced collagen production. Initially, we examined the effect of knocking-down TβRI in stellate cells on collagen production. Stellate cells were transfected with control siRNA (siCtl) or TβRI siRNA (siTβRI), allowed to recover and then treated with Vector-and MT1-MMP-conditioned media. As shown in Fig. 6B, siTβRI successfully knocked-down TβRI protein expression and attenuated the effect of MT1-MMP-conditioned media on collagen mRNA expression by stellate cells. We also used a well-characterized small molecule inhibitor of TβRI kinase activity (SB431542, Supplemental Fig. S4B) on MT1-MMP-conditioned media-induced collagen production. As shown in Fig. 6C, SB431542 attenuated the effect of MT1-MMP-conditioned media on collagen mRNA expression by stellate cells. We also used a function blocking anti-TGF-β1-3 antibody (Supplemental Fig. S4B) to determine whether the increased TGF-β signaling and collagen expression by stellate cells was due to increased active TGF-β levels in the MT1-MMP-conditioned media. Vector- and MT1-MMP-conditioned media from AsPC1 and Panc1 cells were pre-incubated with either control IgG antibody or anti-TGF-β antibody for 30 min and then the conditioned media were added to stellate cells to examine the effect on collagen mRNA expression. As shown in Fig. 6D, MT1-MMP-conditioned media pre-incubated with control IgG induced collagen mRNA expression in stellate cells, while the conditioned media pre-incubated with anti-TGF-β antibody failed to induce collagen mRNA expression in stellate cells. We also examined TGF-β1, -β2, and -β3 mRNA levels by qRT-PCR from AsPC1- and Panc1-Vector and -MT1-MMP cells grown in collagen to determine if there was increased production of TGF-β affecting stellate cells. There was no pronounced difference in the TGF-β isotypes as a result of MT1-MMP expression in Panc1 and AsPC1 cells (Supplemental Fig. S4C). Since the function-blocking anti-TGF-β antibody blocks stellate cell collagen production (Fig. 6C), these results overall suggest that the affect on the stellate cells from MT1-MMP expression in pancreatic cancer cell is primarily due to increased levels of the active form of TGF-β.

DISCUSSION

MT1-MMP plays an essential role in tumor progression, especially in the collagen-rich tumor microenvironment (19, 22, 23, 25). Although MT1-MMP-null mice have been generated, these mice die within 2-3 months of age because of severe skeletal and soft tissue abnormalities (20, 21). Consequently, it was not possible to examine the role of MT1-MMP in PDAC development by crossing MT1-MMP-null mice to the now well-defined mouse models of PDAC (32, 33, 35, 36). Thus, we created mice that overexpress human MT1-MMP in the mouse pancreas; however, MT1-MMP expression alone was not sufficient to induce any phenotypic changes. It is now well established that an activating mutation of Kras is often necessary to generate phenotypic changes in the mouse pancreas (31, 37). Several studies have shown that modulation of the TGF-β pathway, for example through selective deletion of Smad4 or loss of TβRII in the mouse pancreas, by itself showed no discernible phenotype. However, disrupting this pathway in combination with activated KrasG12D resulted in development of PDAC tumors (32, 35, 36, 38). Interestingly, MT1-MMP expression in the mouse mammary gland also failed to develop any phenotypic changes in nulliparous mice and the effect of MT1-MMP was only seen in multiparous mice (23), further demonstrating that MT1-MMP alone is not sufficient to cause gross phenotypic changes.

Although we used the elastase promoter to target Kras to acinar cells, until recently it was debated whether the EL-Kras mouse model represented an appropriate model to study human disease. A 2006 review of all mouse models concluded that EL-Kras, while showing evidence of cystic papillary neoplasms and mouse PanIN-like (mPanIN) lesions arising in the background of ADM, did not recapitulate the human disease as well as other models. The primary contention was that the ductal abnormalities seen, while resembling mPanINs, arose only in the setting of ADM (39). However, three different groups recently clearly demonstrated that all cell lineages within the pancreas could give rise to ductal neoplasms that model human disease (40-42). One group showed that targeting Kras to endocrine cells that are normally refractory to transformation by oncogenes can, in the setting of chronic pancreatic injury, lead to the development of PDAC that closely recapitulates human disease (40). Two other groups showed that targeting Kras to acinar cells leads to the development of ductal mPanINs with nearly identical features to those that develop from broad embryonic targeting of the pancreas using a Pd×1 promoter (41, 42). These findings suggest that ductal abnormalities arising within areas of metaplasia, such as in our EL-Kras model, are valid models to study human disease.

Morphologically, MT1-MMP expression in the KrasG12D background caused lesions similar to human IPMNs (34). Although most transgenic mouse models of pancreatic cancer develop PanIN precursor lesions that progress to invasive disease, some of the transgenic mouse models develop IPMNs (32, 35). Two groups reported that loss of Smad4 in the KrasG12D background causes IPMNs in the majority of mice (32, 35). Even though it is not clear why MT1-MMP expression results in lesions with mucin production, it is interesting to note that MT1-MMP in the airways can induce a phenotypic shift to increase mucin production and can also proteolytically cleave the membrane bound mucins from the surface of epithelial cells (43, 44).

The lesions that developed in the MT1-MMP-expressing mice demonstrated significant fibrosis. Although the mechanism was not identified, it was previously noted that expression of MT1-MMP in mouse mammary gland resulted in significant fibrosis (23), while expression of MT1-MMP in MDCK and squamous cell cancer cells caused significant fibrosis when the cells were injected in vivo (23-25). We show here that the increased fibrosis was associated with increased Smad2 phosphorylation, indicating increased TGF-β signaling in MT1-MMP-expressing mice. The association between MT1-MMP and p-Smad2 has been seen in human pancreatic cancer specimens as well. In a human pancreatic cancer tissue microarray MT1-MMP expression was increased in areas of fibrosis, as was the amount of p-Smad2 (11). In cardiac tissue, forced expression MT1-MMP in cardiac tissue caused significant fibrosis that was also associated with increased Smad2 phosphorylation (45).

Significantly, in this report we identified a potential mechanism by which MT1-MMP induces fibrosis. The lesions we observed were associated with increased α-SMA(+) cells, consistent with increased numbers of myofibroblasts in the pancreatic tumors. Using an in vitro model, we showed that MT1-MMP expression in PDAC cells increased pancreatic stellate cell collagen production through increased TGF-β signaling, indicating cross-talk between cancer cells and stellate cells involving MT1-MMP and TGF-β to promote fibrosis. Previously, it was shown that MT1-MMP can increase the levels of the active-form of TGF-β by cleaving the latent TGF-β binding protein-1 to release TGF-β bound to the ECM, increasing the biological availability of TGF-β (46-48). MT1-MMP can also cleave the latency-associated peptide to release the active-form of TGF-β (45, 48-50). In our in vitro model, we found that the MT1-MMP-conditioned media rapidly induced Smad2 phosphorylation and that function-blocking anti-TGF-β antibody significantly abrogated MT1-MMP-conditioned media-induced stellate cell collagen production, demonstrating that MT1-MMP expression in PDAC cells grown in 3D collagen increases the level of the active-form of TGF-β.

Overall, we demonstrate that MT1-MMP cooperates with KrasG12D to increase the number and size of dysplastic precancerous lesions, the extent of TGF-β signaling within the lesions and the surrounding stroma, and the amount of fibrosis surrounding each lesion. Mechanistically, MT1-MMP increases the amount of active-form of TGF-β within the microenvironment, activating pancreatic stellate cells and inducing production of more type I collagen. Given that MT1-MMP promotes tumor growth along with significantly increased tumor fibrosis, selective targeting of MT1-MMP may not only limit PDAC progression, but may also attenuate fibrosis. As a recent report has established that fibrosis limits delivery and efficacy of chemotherapy in pancreatic cancer (5), it will be important to define whether targeting MT1-MMP can enhance the response to current therapeutic modalities by attenuating fibrosis.

Supplementary Material

1

ACKNOWLEDGEMENTS

This research was supported by grant R01CA126888 (H.G.M.) from the NCI, the National Pancreas Foundation (H.G.M.), the Rosenberg Family Foundation (H.G.M.) the Association for Academic Surgery Foundation (S.B.K), the Nathan and Isabel Miller Family Foundation (D.J.B.), the IDP Foundation (D.J.B.), and an AACR-Pancreatic Cancer Action Network Career Development Award (P.J.G.).

REFERENCES

  • 1.Jemal A, Siegel R, Ward E, Hao Y, Xu J, Thun MJ. Cancer statistics, 2009. CA Cancer J Clin. 2009;59:225–49. doi: 10.3322/caac.20006. [DOI] [PubMed] [Google Scholar]
  • 2.Chu GC, Kimmelman AC, Hezel AF, Depinho RA. Stromal biology of pancreatic cancer. J Cell Biochem. 2007;101:887–907. doi: 10.1002/jcb.21209. [DOI] [PubMed] [Google Scholar]
  • 3.Keleg S, Büchler P, Ludwig R, Büchler MW, Friess H. Invasion and metastasis in pancreatic cancer. Mol Cancer. 2003;2:14. doi: 10.1186/1476-4598-2-14. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Hidalgo M. Pancreatic cancer. N Engl J Med. 2010;362:1605–17. doi: 10.1056/NEJMra0901557. [DOI] [PubMed] [Google Scholar]
  • 5.Olive KP, Jacobetz MA, Davidson CJ, Gopinathan A, McIntyre D, Honess D, et al. Inhibition of Hedgehog signaling enhances delivery of chemotherapy in a mouse model of pancreatic cancer. Science. 2009;324:1457–61. doi: 10.1126/science.1171362. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Radisky DC, Kenny PA, Bissell MJ. Fibrosis and cancer: do myofibroblasts come also from epithelial cells via EMT? J Cell Biochem. 2007;101:830–9. doi: 10.1002/jcb.21186. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Orimo A, Gupta PB, Sgroi DC, Arenzana-Seisdedos F, Delaunay T, Naeem R, et al. Stromal fibroblasts present in invasive human breast carcinomas promote tumor growth and angiogenesis through elevated SDF-1/CXCL12 secretion. Cell. 2005;121:335–48. doi: 10.1016/j.cell.2005.02.034. [DOI] [PubMed] [Google Scholar]
  • 8.Yen TWf, Aardal NP, Bronner MP, Thorning DR, Savard CE, Lee SP, et al. Myofibroblasts are responsible for the desmoplastic reaction surrounding human pancreatic carcinomas. Surgery. 2002;131:129–34. doi: 10.1067/msy.2002.119192. [DOI] [PubMed] [Google Scholar]
  • 9.Hwang RF, Moore T, Arumugam T, Ramachandran V, Amos KD, Rivera A, et al. Cancer-associated stromal fibroblasts promote pancreatic tumor progression. Cancer Res. 2008;68:918–26. doi: 10.1158/0008-5472.CAN-07-5714. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Vonlaufen A, Phillips PA, Xu Z, Goldstein D, Pirola RC, Wilson JS, et al. Pancreatic stellate cells and pancreatic cancer cells: an unholy alliance. Cancer Res. 2008;68:7707–10. doi: 10.1158/0008-5472.CAN-08-1132. [DOI] [PubMed] [Google Scholar]
  • 11.Ottaviano AJ, Sun L, Ananthanarayanan V, Munshi HG. Extracellular matrix-mediated membrane-type 1 matrix metalloproteinase expression in pancreatic ductal cells is regulated by transforming growth factor-beta1. Cancer Res. 2006;66:7032–40. doi: 10.1158/0008-5472.CAN-05-4421. [DOI] [PubMed] [Google Scholar]
  • 12.Friess H, Yamanaka Y, Büchler M, Ebert M, Beger HG, Gold LI, et al. Enhanced expression of transforming growth factor beta isoforms in pancreatic cancer correlates with decreased survival. Gastroenterology. 1993;105:1846–56. doi: 10.1016/0016-5085(93)91084-u. [DOI] [PubMed] [Google Scholar]
  • 13.Apte MV, Park S, Phillips PA, Santucci N, Goldstein D, Kumar RK, et al. Desmoplastic reaction in pancreatic cancer: role of pancreatic stellate cells. Pancreas. 2004;29:179–87. doi: 10.1097/00006676-200410000-00002. [DOI] [PubMed] [Google Scholar]
  • 14.Aoyagi Y, Oda T, Kinoshita T, Nakahashi C, Hasebe T, Ohkohchi N, et al. Overexpression of TGF-beta by infiltrated granulocytes correlates with the expression of collagen mRNA in pancreatic cancer. Br J Cancer. 2004;91:1316–26. doi: 10.1038/sj.bjc.6602141. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Sato H, Takino T, Okada Y, Cao J, Shinagawa A, Yamamoto E, et al. A matrix metalloproteinase expressed on the surface of invasive tumour cells. Nature. 1994;370:61–5. doi: 10.1038/370061a0. [DOI] [PubMed] [Google Scholar]
  • 16.Okada A, Bellocq JP, Rouyer N, Chenard MP, Rio MC, Chambon P, et al. Membrane-type matrix metalloproteinase (MT-MMP) gene is expressed in stromal cells of human colon, breast, and head and neck carcinomas. P Natl Acad Sci USA. 1995;92:2730–4. doi: 10.1073/pnas.92.7.2730. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Iacobuzio-Donahue CA, Ryu B, Hruban RH, Kern SE. Exploring the host desmoplastic response to pancreatic carcinoma: gene expression of stromal and neoplastic cells at the site of primary invasion. Am J Pathol. 2002;160:91–9. doi: 10.1016/S0002-9440(10)64353-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Bramhall SR, Neoptolemos JP, Stamp GW, Lemoine NR. Imbalance of expression of matrix metalloproteinases (MMPs) and tissue inhibitors of the matrix metalloproteinases (TIMPs) in human pancreatic carcinoma. J Pathol. 1997;182:347–55. doi: 10.1002/(SICI)1096-9896(199707)182:3<347::AID-PATH848>3.0.CO;2-J. [DOI] [PubMed] [Google Scholar]
  • 19.Imamura T, Ohshio G, Mise M, Harada T, Suwa H, Okada N, et al. Expression of membrane-type matrix metalloproteinase-1 in human pancreatic adenocarcinomas. J Cancer Res Clin Oncol. 1998;124:65–72. doi: 10.1007/s004320050137. [DOI] [PubMed] [Google Scholar]
  • 20.Zhou Z, Apte SS, Soininen R, Cao R, Baaklini GY, Rauser RW, et al. Impaired endochondral ossification and angiogenesis in mice deficient in membrane-type matrix metalloproteinase I. P Natl Acad Sci USA. 2000;97:4052–7. doi: 10.1073/pnas.060037197. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Holmbeck K, Bianco P, Caterina J, Yamada S, Kromer M, Kuznetsov SA, et al. MT1-MMP-deficient mice develop dwarfism, osteopenia, arthritis, and connective tissue disease due to inadequate collagen turnover. Cell. 1999;99:81–92. doi: 10.1016/s0092-8674(00)80064-1. [DOI] [PubMed] [Google Scholar]
  • 22.Hotary KB, Allen ED, Brooks PC, Datta NS, Long MW, Weiss SJ. Membrane type I matrix metalloproteinase usurps tumor growth control imposed by the three-dimensional extracellular matrix. Cell. 2003;114:33–45. doi: 10.1016/s0092-8674(03)00513-0. [DOI] [PubMed] [Google Scholar]
  • 23.Ha HY, Moon HB, Nam MS, Lee JW, Ryoo ZY, Lee TH, et al. Overexpression of membrane-type matrix metalloproteinase-1 gene induces mammary gland abnormalities and adenocarcinoma in transgenic mice. Cancer Res. 2001;61:984–90. [PubMed] [Google Scholar]
  • 24.Dangi-Garimella S, Redig AJ, Shields MA, Siddiqui MA, Munshi HG. Rho-ROCK-myosin signaling mediates membrane type 1 matrix metalloproteinase-induced cellular aggregation of keratinocytes. J Biol Chem. 2010;285:28363–72. doi: 10.1074/jbc.M110.146019. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Soulié P, Carrozzino F, Pepper MS, Strongin AY, Poupon M-F, Montesano R. Membrane-type-1 matrix metalloproteinase confers tumorigenicity on nonmalignant epithelial cells. Oncogene. 2005;24:1689–97. doi: 10.1038/sj.onc.1208360. [DOI] [PubMed] [Google Scholar]
  • 26.Grippo PJ, Nowlin PS, Demeure MJ, Longnecker DS, Sandgren EP. Preinvasive pancreatic neoplasia of ductal phenotype induced by acinar cell targeting of mutant Kras in transgenic mice. Cancer Res. 2003;63:2016–9. [PubMed] [Google Scholar]
  • 27.Guerra C, Schuhmacher AJ, Cañamero M, Grippo PJ, Verdaguer L, Pérez-Gallego L, et al. Chronic pancreatitis is essential for induction of pancreatic ductal adenocarcinoma by K-Ras oncogenes in adult mice. Cancer Cell. 2007;11:291–302. doi: 10.1016/j.ccr.2007.01.012. [DOI] [PubMed] [Google Scholar]
  • 28.Strouch MJ, Cheon EC, Salabat MR, Krantz SB, Gounaris E, Melstrom LG, et al. Crosstalk between mast cells and pancreatic cancer cells contributes to pancreatic tumor progression. Clin Cancer Res. 2010;16:2257–65. doi: 10.1158/1078-0432.CCR-09-1230. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Diamond ME, Sun L, Ottaviano AJ, Joseph MJ, Munshi HG. Differential growth factor regulation of N-cadherin expression and motility in normal and malignant oral epithelium. J Cell Sci. 2008;121:2197–207. doi: 10.1242/jcs.021782. [DOI] [PubMed] [Google Scholar]
  • 30.Klimstra DS, Longnecker DS. K-ras mutations in pancreatic ductal proliferative lesions. Am J Pathol. 1994;145:1547–50. [PMC free article] [PubMed] [Google Scholar]
  • 31.Almoguera C, Shibata D, Forrester K, Martin J, Arnheim N, Perucho M. Most human carcinomas of the exocrine pancreas contain mutant c-K-ras genes. Cell. 1988;53:549–54. doi: 10.1016/0092-8674(88)90571-5. [DOI] [PubMed] [Google Scholar]
  • 32.Bardeesy N, Cheng K-H, Berger JH, Chu GC, Pahler J, Olson P, et al. Smad4 is dispensable for normal pancreas development yet critical in progression and tumor biology of pancreas cancer. Genes Dev. 2006;20:3130–46. doi: 10.1101/gad.1478706. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Izeradjene K, Combs C, Best M, Gopinathan A, Wagner A, Grady WM, et al. Kras(G12D) and Smad4/Dpc4 haploinsufficiency cooperate to induce mucinous cystic neoplasms and invasive adenocarcinoma of the pancreas. Cancer Cell. 2007;11:229–43. doi: 10.1016/j.ccr.2007.01.017. [DOI] [PubMed] [Google Scholar]
  • 34.Hruban RH, Takaori K, Klimstra DS, Adsay NV, Albores-Saavedra J, Biankin AV, et al. An illustrated consensus on the classification of pancreatic intraepithelial neoplasia and intraductal papillary mucinous neoplasms. Am J Surg Pathol. 2004;28:977–87. doi: 10.1097/01.pas.0000126675.59108.80. [DOI] [PubMed] [Google Scholar]
  • 35.Kojima K, Vickers SM, Adsay NV, Jhala NC, Kim H-G, Schoeb TR, et al. Inactivation of Smad4 accelerates Kras(G12D)-mediated pancreatic neoplasia. Cancer Res. 2007;67:8121–30. doi: 10.1158/0008-5472.CAN-06-4167. [DOI] [PubMed] [Google Scholar]
  • 36.Rustgi AK. The molecular pathogenesis of pancreatic cancer: clarifying a complex circuitry. Genes Dev. 2006;20:3049–53. doi: 10.1101/gad.1501106. [DOI] [PubMed] [Google Scholar]
  • 37.Hezel AF, Kimmelman AC, Stanger BZ, Bardeesy N, Depinho RA. Genetics and biology of pancreatic ductal adenocarcinoma. Genes Dev. 2006;20:1218–49. doi: 10.1101/gad.1415606. [DOI] [PubMed] [Google Scholar]
  • 38.Ijichi H, Chytil A, Gorska AE, Aakre ME, Fujitani Y, Fujitani S, et al. Aggressive pancreatic ductal adenocarcinoma in mice caused by pancreas-specific blockade of transforming growth factor-beta signaling in cooperation with active Kras expression. Genes Dev. 2006;20:3147–60. doi: 10.1101/gad.1475506. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Hruban RH, Adsay NV, Albores-Saavedra J, Anver MR, Biankin AV, Boivin GP, et al. Pathology of genetically engineered mouse models of pancreatic exocrine cancer: consensus report and recommendations. Cancer Res. 2006;66:95–106. doi: 10.1158/0008-5472.CAN-05-2168. [DOI] [PubMed] [Google Scholar]
  • 40.Gidekel Friedlander SY, Chu GC, Snyder EL, Girnius N, Dibelius G, Crowley D, et al. Context-dependent transformation of adult pancreatic cells by oncogenic K-Ras. Cancer Cell. 2009;16:379–89. doi: 10.1016/j.ccr.2009.09.027. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41.De La OJ-P, Emerson LL, Goodman JL, Froebe SC, Illum BE, Curtis AB, et al. Notch and Kras reprogram pancreatic acinar cells to ductal intraepithelial neoplasia. P Natl Acad Sci USA. 2008;105:18907–12. doi: 10.1073/pnas.0810111105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42.Habbe N, Shi G, Meguid RA, Fendrich V, Esni F, Chen H, et al. Spontaneous induction of murine pancreatic intraepithelial neoplasia (mPanIN) by acinar cell targeting of oncogenic Kras in adult mice. P Natl Acad Sci USA. 2008;105:18913–8. doi: 10.1073/pnas.0810097105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43.Deshmukh HS, McLachlan A, Atkinson JJ, Hardie WD, Korfhagen TR, Dietsch M, et al. Matrix metalloproteinase-14 mediates a phenotypic shift in the airways to increase mucin production. Am J Respir Crit Care. 2009;180:834–45. doi: 10.1164/rccm.200903-0328OC. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44.Thathiah A, Carson DD. MT1-MMP mediates MUC1 shedding independent of TACE/ADAM17. Biochem J. 2004;382:363–73. doi: 10.1042/BJ20040513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 45.Spinale FG, Escobar GP, Mukherjee R, Zavadzkas JA, Saunders SM, Jeffords LB, et al. Cardiac-restricted overexpression of membrane type-1 matrix metalloproteinase in mice: effects on myocardial remodeling with aging. Circ Heart Fail. 2009;2:351–60. doi: 10.1161/CIRCHEARTFAILURE.108.844845. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 46.Karsdal MA, Larsen L, Engsig MT, Lou H, Ferreras M, Lochter A, et al. Matrix metalloproteinase-dependent activation of latent transforming growth factor-beta controls the conversion of osteoblasts into osteocytes by blocking osteoblast apoptosis. J Biol Chem. 2002;277:44061–7. doi: 10.1074/jbc.M207205200. [DOI] [PubMed] [Google Scholar]
  • 47.Freudenberg J, Chen W. Induction of Smad1 by MT1-MMP contributes to tumor growth. Int J Cancer. 2007;121:966–77. doi: 10.1002/ijc.22754. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 48.Kessenbrock K, Plaks V, Werb Z. Matrix metalloproteinases: regulators of the tumor microenvironment. Cell. 2010;141:52–67. doi: 10.1016/j.cell.2010.03.015. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 49.Tatti O, Vehviläinen P, Lehti K, Keski-Oja J. MT1-MMP releases latent TGF-beta1 from endothelial cell extracellular matrix via proteolytic processing of LTBP-1. Exp Cell Res. 2008;314:2501–14. doi: 10.1016/j.yexcr.2008.05.018. [DOI] [PubMed] [Google Scholar]
  • 50.Böttinger EP, Factor VM, Tsang ML, Weatherbee JA, Kopp JB, Qian SW, et al. The recombinant proregion of transforming growth factor beta1 (latency-associated peptide) inhibits active transforming growth factor beta1 in transgenic mice. P Natl Acad Sci USA. 1996;93:5877–82. doi: 10.1073/pnas.93.12.5877. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

1
NIHMS320257-supplement-1.pdf (6MB, pdf)

RESOURCES