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Logo of jbcThe Journal of Biological Chemistry
 
J Biol Chem. 2012 September 21; 287(39): 32467–32478.
Published online 2012 August 2. doi:  10.1074/jbc.M112.370064
PMCID: PMC3463308

Overexpression and β-1,6-N-Acetylglucosaminylation-initiated Aberrant Glycosylation of TIMP-1

A “DOUBLE WHAMMY” STRATEGY IN COLON CANCER PROGRESSION*An external file that holds a picture, illustration, etc.
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Abstract

There has been ongoing debate over whether tissue inhibitor of metalloproteinase-1 (TIMP-1) is pro- or anti-oncogenic. We confirmed that TIMP-1 reinforced cell proliferation in an αvβ3 integrin-dependent manner and conferred resistance against cytotoxicity triggered by TNF-α and IL-2 in WiDr colon cancer cells. The cell-proliferative effects of TIMP-1 contributed to clonogenicity and tumor growth during the onset and early phase of tumor formation in vivo and in vitro. However, mass-produced TIMP-1 impeded further tumor growth by tightly inhibiting the activities of collagenases, which are critical for tumor growth and malignant transformation. Tumor cells could overcome this impasse by overexpression of N-acetylglucosaminyltransferase V, which deteriorates TIMP-1 into an aberrant glycoform. The aberrant glycoform of TIMP-1 was responsible for the mitigated inhibition of collagenases. The outbalanced activities of collagenases can degrade the basement membrane and the interstitial matrix, which act as a physical barrier for tumor growth and progression more efficiently. The concomitant overexpression of TIMP-1 and N-acetylglucosaminyltransferase V enabled WiDr cells to show a higher tumor growth rate as well as more malignant behaviors in a three-dimensional culture system.

Keywords: Colon Cancer, Glycosylation, Glycosyltransferases, Tissue Inhibitors of Metalloprotease (TIMPs), Tumor Microenvironment, Aberrant Glycosylation, GnT-V, TIMP-1

Introduction

Glycosylation is an enzymatic modification that is commonly found in eukaryotic cells, and more than 50% of eukaryotic proteins are estimated to be glycosylated (1). Glycosylation is critical for many biological processes, including cell-cell or cell-extracellular matrix (ECM)3 communication (2). In particular, membrane and secreted proteins play roles as glycoforms that are otherwise difficult to do (3). Eukaryotic cells have adopted protein glycosylation under evolutionary pressure to fine tune biological processes and maintain cellular stability. However, failures or defects in proper glycosylation lead to various diseases, including congenital disorders of glycosylation (4). Evidence that aberrant glycosylation is frequently found in cancer is rapidly accumulating (5). This implies that transformed cells manipulate glycosylation machinery to create favorable conditions for tumorigenesis and cancer progression. Despite the prevalence of aberrant glycosylation in cancer, mechanistic studies to elucidate the role of aberrant glycosylation at the molecular level have been relatively sparse.

Recently, mounting evidence has highlighted the importance of ECM during cancer development and progression (6, 7). Deregulated ECM dynamics are caused by tumors and interacting stromal cells and non-cellular components and, in turn, allow tumor cells to evolve in the niche (8). This reciprocal communication directs cancer progression. Central to the deregulation of the tumor microenvironment are the matrix metalloproteinases (MMPs), which are tightly controlled under normal conditions, and an imbalance between MMPs and their regulators is observed during cancer progression (9). Tissue inhibitor of metalloproteinase-1 (TIMP-1) is a key regulator for several MMPs and has, at the same time, an MMP-independent cell growth-potentiating function (10). These dual roles of TIMP-1 make it difficult to assess the net contribution of the protein to cancer progression. TIMP-1 has also been shown to enhance the proliferation of a number of transformed cells and tumor cell lines (1114) and confer resistance against apoptotic cues (15, 16). However, in vivo studies indicate an inhibitory function of TIMP-1 during cancer progression (17, 18). In these regards, the roles of TIMP-1 in tumorigenesis and cancer progression are quite contradictory. Moreover, it is puzzling to find that TIMP-1 is accumulated in tumor tissues (19, 20), and the accumulated TIMP-1 is associated with poor prognosis (21).

We here provide evidence that the normally glycosylated form of TIMP-1 promotes tumor cell proliferation and confers resistance against cytotoxicity triggered by cytokines via an MMP-independent mechanism during the early stage of cancer. The antiproteolytic burden created by TIMP-1 accumulation is reduced by aberrant glycosylation initiated by N-acetylglucosaminyltransferase V (GnT-V). In other words, colon cancer cells utilize the growth-potentiating activity of TIMP-1 at the early stage of cancer and employ an aberrant glycosylation mechanism for TIMP-1 to nullify the MMP-dependent, cancer-suppressive effects to support cancer progression. We provide in vivo and in vitro evidence as well as results from a clinical investigation that support this understanding.

EXPERIMENTAL PROCEDURES

Modulation of GnT-V and TIMP-1 Expression in WiDr Cells

GnT-V-overexpressing cells and mock cells were established following transfection of mgat5/pCXN (neo) and the empty vector, respectively, into the parental WiDr cells, a derivative of the human colonic adenocarcinoma cell line HT-29 (22), using Lipofectamine PlusTM reagent (Invitrogen) according to the manufacturer's instructions. A stable clone that overexpresses GnT-V was selected after dozens of subcultures. TIMP-1 mutant genes were generated using the standard megaprimer methods, where Thr32 and Ser80 were mutated to valine and alanine, respectively. Wild type TIMP-1 and the mutant genes were cloned into pcDNA 3.1 hygro(+) plasmid vector (Invitrogen). The cloned vectors were transfected into WiDr:mock or WiDr:GnT-V cells. The stable TIMP-1 transfectants were confirmed by an immunoblot analysis. Cells were maintained as a monolayer in RPMI 1640 medium containing 10% fetal bovine serum at 37 °C, supplied with 5% CO2.

Western and Lectin Blot Analyses

Protein preparations were resolved on 10–15% SDS-polyacrylamide gels and transferred electrically onto PVDF membranes (Immobilon-P, Millipore). The membranes were blocked in 0.05% Tween 20-TBS containing 5% skim milk (immunoblot) or 3% BSA (lectin blot) and then incubated with primary antibodies or biotin-labeled L4-phytohemagglutinin (L4-PHA) or concanavalin A. After hybridizing with HRP-labeled secondary antibody (Cell Signaling) or HRP-avidin conjugates (Vector Laboratories, Inc.), the membranes were reacted with ECLTM Western blotting detection reagents (Amersham Biosciences) and exposed to an x-ray film for 1–2 min. The band intensity was calculated from the digitized, scanned files using ImageJ software (National Institutes of Health, Bethesda, MD).

In Vivo Animal Studies

Six-week-old C.By.Cg-Foxn1nu (nude) mice were housed and maintained in an animal facility under specific pathogen-free conditions with continuous microbiological monitoring. A sterilized commercial diet (Harlan) and water were given ad libitum. Cells (2 × 106 cells in 100 ml of PBS) were injected subcutaneously into the thighs of nude mice. The nude mice (n = 10/group) were inspected daily, and tumor size was measured three times daily in two dimensions with calipers. The tumor volumes were calculated using the formula, length × (width)2/2. For histopathological examination, subcutaneous tumor masses were sectioned from the sacrificed mice and fixed in 10% neutral buffered formalin. After fixation, the specimens were routinely processed, embedded in paraffin, and stained with hematoxylin and eosin.

Wound Healing Assay

Cells were grown to near confluence in 6-well plates and scraped using a 20-μl pipette tip. Floating cells were removed after washing with PBS buffer and grown for 5 days. The scratched regions were photographed daily at a ×200 magnification. Distance filled with grown cells was calculated at different times, and the relative healing velocity was obtained from the regression curves.

Sodium 3′-[1-(Phenylaminocarbonyl)-3,4-tetrazolium]-bis(4-methoxy-6-nitro)benzene Sulfonic Acid Hydrate (XTT) Assay

A cell proliferation assay using XTT reagent purchased from Roche Applied Science was performed according to the manufacturer's instructions. Briefly, cells (1 × 104) were plated in 96-well microplates and grown for 2 days. Cells were incubated with 50 μl of XTT labeling agent and 1 μl of electron coupling agent at 37 °C for 4 h. Absorbance at 495 and 695 nm was measured.

BrdU Incorporation Assay

A total of 1 × 106 cells were incubated with 50 ng/ml tumor necrosis factor (TNF)-α and 0.2 μg/ml IL-2 for 2 days and then pulsed with 20 μm BrdU (Sigma) for 1 h at 37 °C. The cells were then fixed with cold 95% ethanol, washed with PBS, and resuspended in 0.4 mg/ml pepsin in 0.1 n HCl for 30 min at room temperature to release nuclei. The nuclei were pelleted, incubated with 2 n HCl for 30 min, and neutralized with 0.1 m Na2B4O7. Nuclei were washed with PBS containing 0.5% Tween 20 and 0.1% BSA. Nuclei were then incubated with anti-BrdU antibody (BD Biosciences) for 90 min in the dark at room temperature. The nuclei were then stained with FITC-labeled goat anti-mouse antibody (1:50) for 30 min at room temperature in the dark. The nuclei were washed with PBS, incubated with propidium iodide (0.1 mg/ml) and RNase A (10 μg/ml) overnight at 4 °C, and then analyzed using a FACSCalibur flow cytometer (BD Biosciences).

FACS Analysis

Cells were cultured in the presence of 50 ng/ml TNF-α and 0.2 μg/ml IL-2 for 2 days. The treated cells were double-stained with Annexin-V-FITC and propidium iodide (PI) as described elsewhere (23). Fluorescence was detected using a fluorescence-activated cell sorter to analyze necrotic (PI+), non-apoptotic (negative for both dyes), early apoptotic (Annexin+/PI−), and late apoptotic cells (Annexin+/PI+). Fluorescence parameters were gated using unstained control cells, and 10,000 cells were counted for each sample.

In Vitro Collagenolysis Inhibition Assays

Cells were cultured in serum-free RPMI1640 medium in the absence of the pH-indicating dye. The conditioned media were retrieved and concentrated using an Amicon ultracentrifugal filter with a molecular mass cut-off of 10 kDa (Millipore). Equal amounts of proteins were mixed with 8 μm fluorogenic substrate, DABCYL-GABA-PQGL-(EDANS)-AK-NH2 (Calbiochem), at 37 °C in 50 mm Tris-HCl buffer (pH 7.5) containing 150 mm NaCl, 5 mm CaCl2, 0.1 mm ZnCl2, and 0.02% Brij-35. The hydrolysis activity was kinetically measured in an LS 45 luminescence spectrometer (PerkinElmer Life Sciences) at excitation and emission wavelengths of 338 and 495 nm, respectively.

Immunoprecipitation

Cells were suspended in PBS buffer containing 0.02% Nonidet P-40 and sonicated on ice. The cell lysates were cleared by centrifugation at 10,000 × g for 10 min. The protein extracts were subject to immunoprecipitation using an integrin β3 monoclonal antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). The precipitated preparations were used for the subsequent Western blot analysis.

Matrigel-based Three-dimensional Culture

Three-dimensional culture was performed as described elsewhere with minor modifications (24). Briefly, 24-well plates were precoated with 50 μl of Matrigel. Approximately 1,500 cells suspended in 150 μl of Matrigel were seeded onto the precoated plate. RPMI 1640 medium containing 1% (v/v) FBS was covered and replaced every 2 days. If necessary, TNF-α and IL-2 were added at the concentrations aforementioned. The cells were incubated at 37 °C for up to 15 days to allow aggregated spheroids to form. The cell aggregates were periodically taken to calculate the formed tumor size. Tumor size was calculated by comparing the lengths of tumors with a mesh of known lengths on photographs. Cell volume was calculated from the formula as used for in vivo experiments. All assays were performed at least three times, with each assay being tested in triplicate.

Statistical Analysis

Statistical differences between groups were determined by Student's t test. Values of p < 0.05 were taken as significant.

RESULTS

Manipulation of the Expression Level and Glycan Structure of TIMP-1 in Colon Cancer Cells

GnT-V is a Golgi-located enzyme that is widely known to be overexpressed in various malignant solid tumors (25), producing β1,6-N-acetylglucosamine (GlcNAc)-branched N-glycan. WiDr is a colon cancer cell line with a minimal expression of GnT-V (26) into which GnT-V (or MGAT5) gene was forced to be stably overexpressed. L4-PHA is a plant-derived lectin that specifically recognizes a β1,6-GlcNAc branch attached to the core mannose residue of N-glycan. Overexpression of GnT-V was confirmed by immunofluoresce using L4-PHA as a probe. The surfaces of cells with GnT-V overexpression were more reactive to L4-PHA binding, compared with the mock transfectant cells (Fig. 1A). The secreted proteins from cells with GnT-V overexpression were also more reactive, as assessed by a lectin blot analysis using L4-PHA (data not shown).

FIGURE 1.
Establishment of stable transfectant cell lines of GnT-V and TIMP-1. A, mannosyl β1,6-GlcNAc linkages on the cell surfaces were traced by immunofluorescence using biotin-labeled L4-PHA as a probe. Nuclei were stained with DAPI. B, aglycosylated ...

TIMP-1 is a glycoprotein that has two N-linked glycosylation sites, at Asn30 and Asn78, and is known to accumulate in various solid tumors, including colon cancer (21). To identify the role of glycans of TIMP-1 and the alterations in cancer progression, the aglycosylation mutant TIMP-1 gene was generated by changing the codon sequences for Thr32 and Ser80 to those for Val32 and Ala80, respectively (Fig. 1B). The wild type or mutant TIMP-1 gene was transfected into the mock and GnT-V-overexpressing cells to establish cancer cell lines in which the glycan structure and expression level of TIMP-1 are manipulated. TIMP-1 shows a molecular mass of ~28.5 kDa, of which N-glycans at both asparagines account for approximately 8 kDa. The stable transfectant cells were confirmed after more than 30 rounds of subculture by immunoblot and lectin blot analyses (Fig. 1C). The aglycosylated TIMP-1 mutant protein was detected with an estimated molecular mass of 20 kDa in the transfectant cells of both mock and GnT-V-overexpressing cells. L4-PHA-reactive aberrant glycoforms of TIMP-1 were observed predominantly in GnT-V-overexpressing cells. The mutant protein showed a 2.5–2.9 fold expression level, compared with the endogenous TIMP-1. Expression level of the wild type TIMP-1 was increased by ~3-fold for TIMP-1 transfectant cells. Cathepsin X was used as a loading control because expression of cathepsin X does not fluctuate by GnT-V expression (26).

To clarify the role of the expression level of TIMP-1 in colon cancer, small hairpin RNA was forced to express to interfere with the endogenous TIMP-1 expression. The stable clones that show the most efficient suppression of TIMP-1 expression were selected, and TIMP-1 expression was also tested after more than 30 rounds of subculture (Fig. 1D). The relative expression levels of TIMP-1 and the mutant form are shown in Fig. 1E. Total and glycosylated TIMP-1 are separately quantified by the enzyme-linked immunoadsorbent assay (Fig. 1E, solid bars) and immunoblot analysis (open bars), respectively.

Biphasic Effects of TIMP-1 in Tumor Formation and Proliferation in Vivo

To investigate the effects of TIMP-1 in tumor formation and proliferation in WiDr cells, each transfectant cell line was injected subcutaneously into the thighs of nude mice, and the tumor volumes were measured daily up to 60 days after injection (Fig. 2A). Tumor masses began to be detectable from 10 days after injection in the case of tumors with the highest growth rate. Mice were sacrificed 60 days after injection, and the formed tumors were resected from mice for further analysis. Analysis of the tumor growth curves revealed three distinct features. 1) Overall, GnT-V transfectant cells showed an earlier solid tumor formation and a higher tumor growth rate, compared with mock cells. 2) Tumor growth showed a biphasic pattern that can be arbitrarily divided into an early phase (days 0–35) and a later phase (days 35–60), and tumors had a higher growth rate in the late phase than in the early phase. 3) The expression level and the glycosylation status of TIMP-1 affected the tumor proliferation, and they worked differently between the early and late phase (Fig. 2B).

FIGURE 2.
Effects of expression level and glycosylation of TIMP-1 on the tumor formation and growth in vivo. A, the arrow indicates a tumor formed 60 days after injection of hT1wt/GnT-V cells into the femur of a nude mouse. B, after injection of each cell line, ...

Analysis of the tumor growth rate in the early phase revealed that the growth rate was proportional to the secretion level of glycosylated TIMP-1 (Fig. 2C) with a squared correlation coefficient (r2) of ≥0.90 regardless of GnT-V expression. Although GnT-V expression contributed to the higher tumor proliferation rate, the extent of dependence on TIMP-1 level was nearly identical between the mock and GnT-V-overexpressing cells. It is of note that overexpression of aglycosylated TIMP-1 did not affect the tumor growth rate in vivo, indicating that glycosylation is critical for TIMP-1 to exert effects on tumor formation and growth at this time point.

In the late phase of tumor growth, the overall rates increased compared with the early phase, as mentioned above. The most interesting feature in this phase is that overexpression of aglycosylated TIMP-1 resulted in a lower tumor growth rate, compared with the control cells (Fig. 2D). TIMP-1 acts as a biological modulator through an MMP-independent mechanism or an MMP-dependent mechanism (27). Aglycosylated TIMP-1 maintains MMP-inhibitory activity (28), and it is thought that the aglycosylated TIMP-1 blocks the proteolytic activities that are critical for tumor growth in the tumor microenvironment. Notably, the down-regulation of MMP-9 resulted in a slower tumor growth in the late phase than in the early phase (supplemental Fig. 1), indicating the pivotal role of MMP-9 in the late phase of tumor growth in vivo. We previously reported that an aberrant glycoform of TIMP-1 was produced in WiDr cells with GnT-V overexpression, and the aberrant form showed mitigated inhibitory activity for MMP-2 and MMP-9 (26).

Consistent with the previous report, a higher tumor growth rate was observed in cells with GnT-V overexpression. Taken together, these results suggest that tumor formation and growth are dependent on the secretion level of glycosylated TIMP-1, and aberrant glycosylation of TIMP-1 by GnT-V could enable MMPs to outbalance the inhibitory action of TIMP-1, thereby accelerating tumor expansion in the late phase.

TIMP-1 Has Tumor Cell Proliferation Activity

The cell growth-potentiating activity of TIMP-1 was tested in vitro using the XTT assay, which shows better performance than the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay with respect to experimental variation and dynamic range (29). The secretion level of glycosylated TIMP-1 was associated with the proliferation capacity of WiDr and the transfectant cells (Fig. 3A). Overexpression of TIMP-1 potentiated proliferation of both parental and GnT-V-overexpressing cells, whereas down-regulation of TIMP-1 decreased proliferative capacity (p < 0.05). Interestingly, aglycosylated TIMP-1 did not show growth-potentiating activity, which was consistent with the results obtained from the tumor growth curve in vivo (Fig. 2B). These results were observed both in the parental and GnT-V-overexpressing cells. The effect of glycosylated TIMP-1 on the cell proliferation was confirmed by the wound-healing assay. The wound-filling activity was increased by overexpression of glycosylated TIMP-1, whereas aglycosylated TIMP-1 showed no such activity (Fig. 3B). The overall wound-filling rate was higher in GnT-V-overexpressing cells, compared with mock cells. However, dependence of wound-filling activity on glycosylated TIMP-1 level was also observed in GnT-V-overexpressing cells.

FIGURE 3.
The tumor proliferation activity of TIMP-1 in vitro. A, cells (1 × 104) were cultured in microplates for 2 days and incubated with XTT mixtures at 37 °C for 4 h. Absorbance at 495 nm was subtracted by the absorbance value at 695 nm (n ...

The cell proliferative activity of TIMP-1 was validated in a specially devised culture dish. The culture flask was separated into three compartments (A, B, and C) by silicon-based barriers, as illustrated in Fig. 3C. The height of the barrier between A and B compartments was lowered for conditioned culture media to freely diffuse between the compartments. The dish-silicon interfaces were tightly sealed, and the C compartment was isolated from the other compartments. To facilitate the diffusion, the dishes were constantly agitated on an orbital shaker. Cells with knockdown expression of TIMP-1 were plated in the B and C compartments, and mock or transfectant cells were cultured in the A compartment. After cells were stabilized, cells in B and C were subjected to scratching, and their wound healing rate was monitored. As the results indicate, the low wound healing activity of cells with down-regulation of TIMP-1 in B was marginally rescued by mock cells placed in A, whereas a significant recovery of the activity in B was observed by the placement of cells with overexpression of glycosylated TIMP-1 in A, compared with the activity of cells in C. However, overexpression of aglycosylated TIMP-1 in A did not result in such a dramatic rescue of wound healing activity (Fig. 3D). Taken together, these results indicate that the proliferation of cancer cells is dependent on the secretion of glycosylated TIMP-1, and the dependence is also seen for the aberrant glycoform of TIMP-1. All of these effects were identically observed for GnT-V transfectant cells.

TIMP-1 Controls Apoptosis Triggered by TNF-α and IL-2

TNF-α is a pleiotropic inflammatory cytokine and causes necrosis of some types of tumors either alone (30) or in combination with chemotherapeutic agents and other cytokines (31). TNF-α and IL-2 are typical cytokines that tumor cells frequently encounter in a tumor microenvironment, where various stromal cells secrete the cytokines during cancer progression (32, 33), and combinatorial treatment of these cytokines showed an efficacy in several clinical trials (34). However, resistance against the cytotoxic effects of TNF-α creates environments for tumor cells to co-evolve and metastasize in various ways (35).

The effects of TIMP-1 expression on the resistance against the cytotoxic effects by TNF-α and IL-2 were investigated by monitoring apoptotic and necrotic features of WiDr and the transfectant cells upon treatment with the cytokines. WiDr cells express TNF-α receptor 1 (TNF-R1), and transfection of the MGAT5 gene did not alter the receptor expression (Fig. 4A). TIMP-1 expression did not affect the expression levels of the receptors for TNF-α and IL-2 (data not shown). Moreover, their receptors were not attacked by GnT-V enzyme. Although treatment with either TNF-α or IL-2 did not show any indication of cytotoxicity as assessed by flow cytometry, the concomitant treatment with 50 ng/ml TNF-α and 200 ng/ml IL-2 triggered both apoptotic and necrotic responses in WiDr and the transfectant cells. However, mock cells were more vulnerable to the cytotoxicity by the cytokines, compared with WiDr/GnT-V cells (Fig. 4B), indicating the cytokine-induced resistance by GnT-V via a yet to be fully defined mechanism independent of TIMP-1. However, the resistance by GnT-V was augmented by the high TIMP-1 secretion (Fig. 4C). Down-regulation of TIMP-1 by RNA interference reversed the apoptotic resistance, confirming that TIMP-1 secretion contributes to avoidance of the cytotoxic effect induced by a combination of cytokines that is encountered by cancer cells in the tumor microenvironment and act as one of the barriers to tumor cells fulfilling metastasis.

FIGURE 4.
Effects of TIMP-1 on resistance against cytotoxicity triggered by TNF-α and IL-2. A, expression of TNF-R1 was investigated by an immunoblot analysis in mock and GnT-V transfectant cells. β-Actin was used as a loading control. B, treated ...

TIMP-1 Binding to αvβ3 Integrin Potentiates Proliferation and Survival Signaling

The molecular mechanism underlying the anti-apoptotic and proliferative effects of TIMP-1 was investigated. Integrin β3 was immunoprecipitated using an anti-integrin β3 antibody, and the binding counterpart integrin αv was found to be co-immunoprecipitated (Fig. 5A). It is of note that TIMP-1 was also co-immunoprecipitated in a TIMP-1 level-dependent manner. Interestingly, TIMP-1 from GnT-V-overexpressing cells appeared to readily bind to the integrin dimer, and the aglycosylated TIMP-1 did not show any interaction with αvβ3 integrin, which may, at least in part, explain why GnT-V confers higher cell proliferation and survival effects and the aglycosylated TIMP-1 has no cell-proliferative activity, as is seen Figs. 224. When treated with TNF-α and IL-2, cells showed the caspase-8-dependent apoptotic death, and only the glycosylated TIMP-1 showed a preventive effect on the caspase-8 activation (Fig. 5B). Likewise, the glycosylated TIMP-1 served to maintain ERK activation even under the apoptotic atmosphere.

FIGURE 5.
Reinforcement of αvβ3 integrin survival and proliferation signaling by TIMP-1. A, TIMP-1 was co-immunoprecipitated (IP) with αvβ3 integrin in a TIMP-1 level-dependent manner. The heavily glycosylated TIMP-1 interacted more ...

Down-regulation of TIMP-1 was responsible for the sensitization of ERK inactivation induced by TNF-α and IL-2 without affecting to the expression of αvβ3 integrin (Fig. 5C). The sensitized ERK inactivation resulted in the down-regulation of cyclin D1 and cyclin A, whose activities are required for cell cycle G1/S transition. In line with this observation, down-regulation of TIMP-1 showed preventive effects on G1/S transition in GnT-V overexpression cells as accessed by a BrdU incorporation assay (Fig. 5D), which was also observed in mock cells. The involvement of TIMP-1 in regulation of cancer cell survival was confirmed by treatment with a neutralizing antibody of TIMP-1 in the culture media. The concomitant treatment of TNF-α and IL-2 resulted in the decreased fraction of cells in S-phase. However, treatment with a neutralizing antibody for TIMP-1 decreased the fraction of cells in S-phase both in the presence and absence of TNF-α and IL-2 (Fig. 5E). Overexpression of TIMP-1 rescued the treated cells from the resting states, inducing transition into the synthetic phase. Taken together, these results indicate that the glycosylated form of TIMP-1 binds to αvβ3 integrin and augments the survival and proliferation signals by the integrin. The heavily glycosylated TIMP-1 from GnT-V-overexpressing cells was found to more readily bind to the integrin molecules. However, determination of whether TIMP-1 binds directly to the integrin molecules or their interaction is mediated by other molecules, such as galectins, awaits further scrutiny.

Effect of Aberrant Glycosylation of TIMP-1 on the Collagenolytic Activity

Cancer cell proliferation was reported to decrease in MMP-9-deficient mice compared with wild type mice (36), and a similar result was obtained in our observations in vivo (supplemental Fig. 1). Moreover, reduced metastasis and angiogenesis of melanoma have been observed in mice that were genetically modified to lack MMP-2 expression (37), indicating the critical role of collagenolytic activity in tumor progression. A histopathological analysis of a tumor formed 60 days after subcutaneous injection indicates that the tumor cells, bordered with dashed lines in Fig. 6A, exhibited an invasive property whereby they proliferate by infiltrating skeletal muscle fibers (filled arrow) and the surrounding endomysium (open arrow), the major component of which is wisps of collagen. The smaller tumors showed lower invasiveness with less vasculature and lower penetration (data not shown). Taken together, these results are suggestive that the net collagenolytic activities are a critical factor for cancer invasion in the cancer-stromal niche.

FIGURE 6.
Mitigated collagenolytic activity of cells with expression of aberrantly glycosylated TIMP-1. A, a tumor formed in a nude mouse was fixed, sliced, and stained with hematoxylin and eosin. The dashed line indicates the tumor cells infiltrating muscle fibers ...

One of the factors governing the invasive potential is the balance between collagenolytic potentials and the inhibitory action by TIMPs. MMP-2 and MMP-9 are major matrix metalloproteinases responsible for deregulation of the ECM. Because the proteases and TIMP-1 are secreted by active tumor cells, the conditioned media, at least partly, reflect the balance between the collagenolytic activity and the inhibitory potential and thus the invasive potential. As an indicator of this, the balanced collagenolytic activity of the conditioned media was measured in vitro. Each cell line was cultured in serum-free media in the absence of dye, and the conditioned media were retrieved. Equal amounts of proteins were mixed with a fluorogenic substrate, and the hydrolysis activity was kinetically measured in a fluorospectrophotometer. The slope of formation of fluorescent products was used to determine the relative collagenolytic activity (Fig. 6B). The kinetic results indicate that cells with high GnT-V expression showed higher collagenolytic activity, compared with the mock cells. Normally glycosylated TIMP-1 substantially inhibited the collagenolytic activity, whereas TIMP-1 down-regulation resulted in elevated activity (Fig. 6C). Of note, aglycosylated TIMP-1 retained inhibition for collagenolysis, which is consistent with a previous report (28). Interestingly, the aberrant TIMP-1 was responsible for the mitigated inhibition of collagenolysis, thereby reinforcing the destructive and invasive potential of cancer cells. Although MMP-2 and MMP-9 were attacked by GnT-V, as assessed by lectin blot analysis using L4-PHA, the difference in the glycoforms did not affect the collagenolytic activity of the two enzymes (supplemental Fig. 2). Taken together, these results suggest that the aberrant glycosylation of TIMP-1 plays a critical role in tumor expansion and regulation of the invasive potential of colon cancer cells. Additionally, other factors that make GnT-V-overexpressing cells more aggressive include the observation that MT1-MMP expression is induced by GnT-V overexpression. As a result, MMP-2 was more readily activated by the membrane-bound MMP, and the free form of active MMP-2 was overproduced (supplemental Fig. 3).

Effects of Aberrant Glycosylation of TIMP-1 on Proliferation in Three-dimensional Culture

It is thought that a monolayer culture system does not mirror the behavior of cancer cells in situ, where cell-ECM interactions are critical for cancer cells to evolve. This prompted us to confirm the results obtained from the monolayered cells in the three-dimensional culture system. We also sought to test if the results obtained in vivo can be reproduced in vitro. For this, cells were embedded in Engelbreth-Holm-Swarm (EHS)-derived matrices as described under “Experimental Procedures.” Type I collagen is the most abundant collagen in the human body, found in the endomysium and interstitial matrix, and acts as a physical barrier for cell migration and interferes with the proliferation of both normal and cancer cells (38). However, EHS-derived matrices do not contain a sufficient amount of type I collagen (38, 39), which we supplemented to EHS-derived matrices at a final concentration of 0.02% (w/v) prior to plating. TNF-α and IL-2 were added beginning at 4 days after seeding to recreate the cytotoxic atmosphere occurring in the tumor environments. Embedded into the matrices, each cell was cultured in low serum conditions (1% FBS, v/v) to enhance the effect of TIMP-1 on tumor cell proliferation. The tumor spheres formed were photographed daily.

As seen in Fig. 7A, a high expression of TIMP-1 contributed to elevating clonogenicity and forming spheres with high tumor volumes. Aberrant glycosylation of TIMP-1 by GnT-V showed a synergistic effect on the tumor expansion, which would otherwise be marginal. When cultured in the presence of excess MMP-2 and MMP-9 inhibitors, cells experienced poor clonogenicity, proliferation, and even an apoptotic death for a portion of cells, indicating the critical role of the MMP activity in tumor formation and progression.

FIGURE 7.
Malignant phenotype and high proliferation of colon cancer cells with concomitant overexpression of GnT-V and TIMP-1 in a three-dimensional culture system. A, embedded in Matrigel supplemented with type I collagen, cells were grown in the presence of ...

Images of tumor spheres that were formed 12 days after seeding clearly illustrate the dependence of clonogenicity and cancer progression on TIMP-1 expression level and aberrant glycosylation of TIMP-1 by GnT-V (Fig. 7B). High expression of TIMP-1 accounted for the higher clone number and voluminous tumor spheres, whereas a portion of cancer cells either showed a lower growth rate or even failed to form a colony in the presence of the cytokines. Aberrant glycosylation of TIMP-1 strengthened the tumor expansion at the late stage. Most interestingly, tumors that concomitantly express TIMP-1 and GnT-V at a high level showed an aggressive morphology with an invasive front (as indicated by the arrows in Fig. 7B), which was difficult to observe in a monolayered cell culture system as well as even in vivo. Cancer cells with marginal expression of GnT-V were symmetrically spherical in shape and appeared to undergo more physical pressure from the ECM as tumors grew. This result is believed to firmly support the claims that the time-dependent interplay of TIMP-1 and GnT-V contribute to tumorigenesis and cancer progression in colon cancer.

DISCUSSION

TIMP-1 is implicated in various biochemical processes by controlling the activities of MMPs and is also known to enhance cell proliferation via an MMP-independent mechanism. These dual activities of TIMP-1 are often contradictory, and the net outcome of TIMP-1 action is complicated and even paradoxical. Especially in cancer, it has not been clearly defined whether TIMP-1 is pro-oncogenic or anti-oncogenic. Many lines of evidence indicate that TIMP-1 promotes cell proliferation and confers resistance against apoptotic cues (40). However, overexpression of TIMP-1, in general, suppresses cancer progression and metastasis in vivo (17, 18), which is also contradictory to the reports that accumulation of TIMP-1 is frequently observed in several types of cancer (19, 20) and correlated with poor prognosis (21).

Here we suggest a plausible model in which TIMP-1 accumulation and subsequent aberrant glycosylation by GnT-V collaborate to direct clonogenicity and cancer progression (supplemental Fig. 4). During the onset of tumor development, it is an urgent matter for a transformed single cell to secure clonogenicity and survive against the cytotoxic and apoptotic signals, including cytokines and immune attacks. The effect of the basement membrane and the interstitial matrix as a physical barrier is negligible at this early stage. It appears that cancer cells utilize the cell-proliferative effect of TIMP-1 to achieve this goal. Overexpression of TIMP-1 positively affected colon cancer cell proliferation (Fig. 3) and resulted in resistance against the cytotoxic effects by concomitant treatments with TNF-α and IL-2 by binding to αvβ3 integrin and reinforcing the survival and proliferation signal (Figs. 4 and and5).5). Actually, overexpression of TIMP-1 culminated in a higher rate of tumor formation and growth at the early stage in vivo (Fig. 2). As tumor growth continues, tumor foci encounter physical barriers by the basement membrane and the interstitial matrix, one of the main components of which is collagens, substrates for MMP-2 and MMP-9. The persistent overexpression of TIMP-1, however, in turn acts as a negative regulator for the collagenolytic enzymes and contributes to pause or retardation of subsequent cancer progression. Notably, the tumor growth was nearly halted in the presence of an excessive concentration of inhibitors for MMP-2 and MMP-9 in the three-dimensional culture system (Fig. 7A). To break this stalemate, cancer cells appear to operate “aberrant glycosylation machinery” by which TIMP-1 deteriorates into a less functional glycoform following the addition of β1,6-GlcNAc to the core N-glycan. β1,6-GlcNAc attached to the core mannose of N-glycan is a preferred substrate for galactosyltransferases and N-acetyltransferases, generating polylactosamine stretch and sialic acid at the terminus (26). This aberrant glycan structure was previously analyzed by a glycan analysis using mass spectrometry, and the aberrant TIMP-1 was found to significantly lose its binding affinity for active MMP-2 and MMP-9 (26). An enhanced collagenolytic activity was observed in WiDr cells with GnT-V overexpression (Fig. 6), and the outbalanced collagenolytic activity was responsible for the subsequent tumor growth in vivo (Fig. 2) and the malignant phenotype in vitro (Fig. 7). Although aglycosylated TIMP-1 is not a form that is found under normal states in nature, it served to support the idea that aberrant glycosylation of TIMP-1 is necessary for accelerated tumor growth. Aglycolsylated TIMP-1 is not attacked by overexpressed GnT-V because of a lack of N-glycan, and the aglycoform retains the collagenase-inhibitory activity (28) (Fig. 6C). Accordingly, cells overexpressing aglycosylated TIMP-1 and GnT-V showed retarded tumor growth in the later phase (Fig. 2).

Many tumors are of epithelium origin and characterized by tight cell-cell adhesion and polarity (41, 42). For tumor cells to obtain invasive and metastatic properties, it is suggested that the epithelial cells need to transform into mesenchymal-like cells, a process called epithelial-mesenchymal transition (EMT). The canonical pathway for EMT is triggered by TGF-β (43), and other pathways have also been reported (4447). Evidence from a number of research groups indicates that several MMPs are involved in EMT. MMP-3 induces EMT in mouse mammary epithelial cells through increased expression of Rac1b and induction of reactive oxygen species (48). MMP-28 (49) and MMP-7 (50) were reported to induce EMT in lung cancer. Activation of MMP-2 and MT-MMP has also been shown to be essential in the EMT of the endocardial cushion cells (51). Tumor cells with an EMT-like phenotype were, although rare, observed in the three-dimensional culture only when GnT-V and TIMP-1 were concomitantly overexpressed (data not shown). Actually, an EMT-like phenotype was observed in GnT-V transgenic mice (52). Because TIMP-1 is a pan-MMP inhibitor (9), overexpression and aberrant glycosylation of TIMP-1 may lead to perturbations in balances with various MMPs that are possibly involved in EMT. Therefore, there is strong motivation to explore which MMP is involved in EMT in colon cancer, whether fluctuations of TIMP-1 affect the MMP-involved EMT, and how collapse in TIMP-1/MMP balance is implicated in EMT.

Cancer cells that form cancer foci dynamically interact with the surrounding stroma that contains fibroblasts, endothelial cells, inflammatory cells, and ECM; participate in the formation of the tumor microenvironment; and evolve into more malignant cells (53). The tumor microenvironment surrounding cancer and stromal cells is best realized in an in vivo model system despite the immunocompromised characteristics in several model mice. Therefore, it is thought that in vivo models provide the native three-dimensional microenvironment and best reflect the behaviors and responses of cancer cells in the tumor environment in situ. However, observations of time course changes in terms of cell morphology are limited, and the final results are attainable only when animals are sacrificed. To overcome this limitation, a three-dimensional in vitro culture system has been employed. Because it is a simplified system and dynamic cellular and chemical interactions are limited, it does not mirror bona fide dynamic behaviors of tumor cells in situ. Here, we attempted to mimic the complex nature of the human tumor microenvironment in a three-dimensional culture system.

We used a laminin-rich matrix derived from EHS as a supporting material. It contains a limited amount of type I collagen, the most abundant collagen in the human body, and acts as a physical barrier for cell migration and interferes with the proliferation of both normal and cancer cells (38). We used an EHS-based matrix supplemented with Type I collagen. Although non-epithelial cells, including immune cells, endothelial cells, and fibroblasts, were not co-cultured, we attempted to establish apoptotic environments by treating TNF-α and IL-2, which occur during interaction of cancer cells with those non-epithelial cells. The concentration of FBS was lowered to 1% (v/v) during three-dimensional culture, with the aim of reflecting the conditions of limited nutrient and molecular factors in the tumor microenvironment. These manipulations were found to suffice to reproduce the in vivo results in this three-dimensional culture system. Deletion of any of these manipulations did not produce in vitro results similar to those obtained from the in vivo xenograft experiments. More comprehensive information on the molecular dynamics, the chemical nature of matrix, and the co-culturing system would help to enhance the three-dimensional culture system such that it could serve as an effective supplemental system for in vivo models and provide information that would otherwise be unobtainable.

Supplementary Material

Supplemental Data:

*This work was supported by the “Convergence Research Center Program” (2010K001302) of the Ministry of Education, Science, and Technology and grants from the Korea Research Institute of Bioscience and Biotechnology Research Initiative Program (KGM3231211).

An external file that holds a picture, illustration, etc.
Object name is sbox.jpgThis article contains supplemental Figs. 1–4.

3The abbreviations used are:

ECM
extracellular matrix
MMP
matrix metalloproteinase
TIMP
tissue inhibitor of metalloproteinase
GnT-V
N-acetylglucosaminyltransferase V
L4-PHA
L4-phytohemagglutinin
PI
propidium iodide
XTT
sodium 3′-[1-(phenylaminocarbonyl)-3,4-tetrazolium]-bis(4-methoxy-6-nitro)benzene sulfonic acid hydrate
TNF-R1
TNF-α receptor 1
EHS
Engelbreth-Holm-Swarm
EMT
epithelial-mesenchymal transition.

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