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[Cancer Research 63, 8312-8317, December 1, 2003]
© 2003 American Association for Cancer Research

Regular Articles

Integrin {alpha}1ß1 and {alpha}2ß1 Are the Key Regulators of Hepatocarcinoma Cell Invasion Across the Fibrotic Matrix Microenvironment

Changqing Yang, Michael Zeisberg, Julie C. Lively, Pia Nyberg, Nezam Afdhal and Raghu Kalluri

Center for Matrix Biology, Division of Gastroenterology, Department of Medicine and The Liver Center, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
As with many types of cancer, cell motility is an important factor in the progression and metastasis of hepatocellular carcinomas (HCC). HCC is associated with significant fibrosis in the liver. The fibrotic microenvironment in the liver is characterized by an altered composition of the extracellular matrix (ECM) and an abundance of growth factors that are likely conducive to migration of HCC cells. The purpose of this study was to delineate promigratory stimuli within the fibrotic microenvironment and to identify specific targets for prevention of HCC cell migration. We used a modified Boyden chamber system that allowed distinction between chemotactic (indirect stimulation) and haptotactic (direct stimulation) migration of two distinct HCC cell lines across the ECM-coated membrane. Fibrotic microenvironment-associated growth factors, such as transforming growth factor ß1 (TGF-ß1), basic fibroblast growth factor (bFGF), and epidermal growth factor (EGF), induced chemotactic and haptotactic migration of HepG2 and Chang cells. Neutralizing antibodies to individual growth factors significantly decreased chemotactic and haptotactic migration. Haptotactic stimulation, but not chemotactic stimulation of HCC cell lines with TGF-ß1, bFGF, and EGF, induced production of matrix metalloproteinase (MMP) 2, a potential mediator of migration. Inhibition of MMPs significantly decreased haptotactic migration induced by individual growth factors but had an insignificant effect on chemotactic migration, suggesting an MMP-independent migration in this setting. Inhibition of cell-ECM interactions with blocking antibodies to {alpha}1 and {alpha}2 integrins were sufficient to inhibit both haptotactic and chemotactic migration induced by individual growth factors, strongly suggesting that targeting these integrins to abrogate pathogenic cell-ECM interactions might be a promising tool for inhibiting growth factor-induced invasion and metastasis of HCC.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
HCC1 is the fifth most common malignancy and is responsible for more than one million deaths annually worldwide (1 , 2) . In most cases, HCC is associated with cirrhosis and fibrosis (2 , 3) . The occurrence of HCC among cirrhotic patients with chronic liver diseases, such as chronic hepatitis B and C or alcohol abuse, is significantly higher (4) . Furthermore, the extent of cirrhosis and fibrosis in patients with HCC is a negative predictor of median survival rate, suggesting that the fibrotic microenvironment is presumably conducive for the progression of HCC (4 , 5) .

Interactions between the host tissue and cancer cells are important for tumor growth and progression, because tumors are dependent on host-tissue derived stromal cells and vasculature for growth and sustenance (6) . Malignant tumor cells recruit stromal cells and vasculature through secretion of growth factors and cytokines, while the host microenvironment contributes to invasiveness and proliferative behavior of cancer cells (7 , 8) . The fibrotic matrix microenvironment differs significantly from the resident ECM, because basement membranes (which provide physical barriers that underlie epithelia and endothelia) are replaced by a less organized fibrillar ECM (which is potentially more permissive for cells to traverse; Refs. 9, 10, 11 ). Within the fibrotic microenvironment, host-derived mesenchymal and mononuclear cells contribute to high levels of profibrotic growth factors, which induce deposition of ECM constituents and matrix-degrading enzymes (12 , 13) .

Local invasion of the host tissue and metastasis are hallmark features of cancer progression (6) . Invasion of tumor cells into the host tissue is regulated by the matrix microenvironment at the tumor-host tissue interface (7 , 14) . Pathological motility of cancer cells plays an important role in both of these events, and understanding of tumor cell motility is important for therapeutic targeting of cancer progression (14) . Migration of cancer cells is likely initiated by cytokines and growth factors, which are released by the host tissue and attract malignant tumor cells to invade the host tissue (15, 16, 17) . Degradation and remodeling of the peri-tumor ECM is considered a necessary step in local tumor invasion (18 , 19) .

Although previous studies have elegantly documented the role of resident ECM in the invasion of HCC cells, the purpose of this study was to investigate the role of the fibrotic microenvironment in the progression of HCC and to identify targets to prevent progression of disease (20, 21, 22, 23, 24) . By using a modified Boyden chamber system that allows delineation of promigratory stimuli present within the fibrotic environment of the cirrhotic liver, we provide evidence that inhibition of pathogenic cell-ECM interactions are potentially a promising target to prevent invasion of HCC cells.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials.
Recombinant human TGF-ß1, EGF, VEGF, and bFGF were purchased from R&D Systems (Minneapolis, MN). Blocking {alpha}1, {alpha}2, and ß1 integrin antibodies were purchased from Chemicon, Inc. (Temecula, CA). Mouse type I and type IV collagens were obtained from Becton Dickinson (Franklin Lakes, NJ). Active MMP-2 enzyme, active MMP-9 enzyme, and monoclonal antibodies to MMP-2 and MMP-9 were purchased from Oncogene, Inc. (Cambridge, MA). Antihuman antibodies for integrin staining were anti-{alpha}1 I domain (FB12), anti-{alpha}2 integrin (P1E6), anti-{alpha}3 integrin (P1B5), and anti-ß1 integrin (6S6) from Chemicon. Secondary antibody was FITC antimouse-IgG (The Jackson Laboratory). Horseradish peroxidase-conjugated secondary antibodies were purchased from DAKO (Glostrup, Denmark).

Cell Culture.
The human hepatocellular cancer cell lines HepG2 and Chang (American Type Culture Collection) were maintained in DMEM or EMEM (Life Technologies, Grand Island, NY) supplemented with 10% FBS, 100 units/ml penicillin, and 100 units/ml streptomycin. The cells were cultured with 5% CO2 and 95% humidity. For experiments, the medium was replaced with DMEM or EMEM without FBS. Although these HCC cells were not from cirrhotic livers, the use of the cells in the context of cirrhotic microenvironment provides an opportunity to assess the influence of matrix microenvironment and cell adhesion on progression of HCC.

Migration Assay.
Migration assays using a Boyden chamber were performed as described previously (10 , 25) . In brief, polyvinyl-pyrrolidone-free polycarbonate membranes with 8-µm pores (Neuro Probes, Inc.) were coated with type IV collagen on the top (50 µg/ml) type I collagen on the bottom (50 µg/ml). The bottom wells of chamber were filled with DMEM or EMEM medium containing supplements according to the specific experimental protocol. Wells were covered with the coated membrane sheet, and 20,000 cells/well, which had been serum-starved for 24 h, were added into the top chamber. The Boyden chamber was incubated for 6 h at 37°C to allow possible migration of cells through the membrane into the bottom chamber. Membranes were stained with Hema3 stain according to manufacturer’s recommendations (Biochemical Sciences, Inc., Swedesboro, NJ). Cells that migrated through the membrane were counted using a counting grid, which was fitted into an eyepiece of a phase contrast microscope. All experiments were repeated at least three times.

Proliferation Assay.
Proliferation assay was performed using [3H]thymidine as described previously (10) . Subconfluent HepG2 or Chang cells were serum-starved for 24 h, trypsinized, and resuspended with DMEM or EMEM containing 0.1% FBS. Cells (3.5 x 105/well) were used in 24-well plates. Plates were incubated for 24 h in a cell incubator. The following day, the medium was replaced with 1 ml medium/0.1% FBS containing TGF-ß1, EGF, VEGF, or bFGF according to the experimental protocol, and the cells were incubated for an additional 12 h. [3H]Thymidine (0.1 microcurie) was added into each well, and the cells were incubated for an additional 12 h before cells were lysed by incubation for 30 min at 60°C in 0.3 M NaOH. The resulting solution was analyzed using a scintillation counter.

Zymography.
HCC cells (2 x 105/well) were plated in 6-well plates and allowed to attach before media were replaced with serum-free DMEM or EMEM, supplemented with growth factors according to the experimental protocol. After 48 h, media were collected and precipitated with ammonium sulfate. The pellet was diluted with PBS, and the solution was dialyzed overnight. Electrophoresis was performed using 15 µg protein solution/lane in 10% gelatin zymogram ready-cast gels (Bio-Rad, Hercules, CA), and active MMP-2 and MMP-9 enzymes were added as control. Gels were washed twice for 20 min at room temperature in renaturing buffer [2.5% (v/v) Triton X-100] and then developed for 18 h at 37°C as described previously (10) . Bands were visualized by Coomassie Blue staining, scanned, and quantified using NIH Image 1.62 software program.

SDS-PAGE Electrophoresis and Western Blotting.
SDS-PAGE electrophoresis and immunoblotting were performed as described previously (10) . In brief, tissue culture supernatants were collected as described for zymography, and 15 µg protein solution/lane were separated by SDS-PAGE electrophoresis and transferred to a nitrocellulose membrane. After blocking, the blots were incubated with antibodies to MMP-2 and MMP-9, washed thoroughly, and incubated with horseradish peroxidase-conjugated secondary antibodies. Antibody binding was visualized by enhanced chemiluminescence (Amersham Biosciences) according to the manufacturer’s protocol.

Flow Cytometry.
Single-cell suspensions were obtained by trypsinization of cultured HepG2 and Chang cells and incubated with 1/100 dilution of the different primary antibodies at 4°C, followed by secondary fluorophore-conjugate antibody incubation at 4°C. Samples were washed twice with PBS/BSA and resuspended in PBS containing Ca2+/Mg2+. Dead cells were excluded by propidium iodide staining. Samples and data were analyzed in a FACScan using CellQuest software (Becton Dickinson, Mountain View, CA).

Statistical Analysis.
Results are expressed as means ± SD. Multiple comparisons were performed by one-way ANOVA in SPSS 10.0 for Windows. P values lower than 0.05 were considered significant.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Schematic Illustration of the Experimental System (Fig. 1)Citation .
We used a two-compartment Boyden chamber that used a polycarbonate membrane coated with type IV collagen and type I collagen (both up-regulated during fibrosis) to delineate regulators of the migration of HepG2 cells (minimally invasive HCC cells) and Chang cells (highly invasive HCC cells) within the fibrotic liver microenvironment interface (7 , 10) . Profibrotic growth factors TGF-ß1, EGF, bFGF, and VEGF, which are abundantly present in the cirrhotic liver microenvironment, were used as direct/haptotactic (represented by addition of growth factors in the top chamber in which the cells are located) and indirect/chemotactic migratory stimuli (represented by addition in the bottom chamber without cells). The role of MMPs in chemotactic and haptotactic migration was assessed by using COL-3 (26 , 27) , a small molecule inhibitor of MMP-2/-3/-9 and of integrin-blocking antibodies (Fig. 1).Citation



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  		Fig. 1. Schematic illustration of the experimental system. A, HepG2 or Chang cells were seeded into the top compartment of a Boyden chamber. A polycarbonate membrane with 8-µm pores, which was coated with type IV collagen on the top and type I collagen on the bottom, separated the top culture chamber from the bottom culture chamber. B, profibrotic growth factors TGF-ß1, EGF, and bFGF were added into the top compartment to induce haptotactic migration. Migration of the HCC cells were assessed by the number of cells that transversed into the bottom chamber in response to haptotactic and chemotactic stimuli. The role of ECM degradation by MMP-2 was evaluated by direct addition of active MMP-2 into the top culture chamber and subsequent MMP-2 inhibition by COL-3. The role of {alpha}1ß1 and {alpha}2ß1 integrins (collagen binding) was determined using integrin-blocking antibodies. C, profibrotic growth factors TGF-ß1, EGF, and bFGF were added into the bottom compartment to induce chemotactic migration. D, without growth factor stimulation, only a few HCC cells migrated through the pores of the membrane and attached to the bottom side of the membrane. E, when the medium in the top compartment was supplemented with TGF-ß1 and EGF, the HCC cells displayed an increased migratory response in an MMP-dependent manner. F, when TGF-ß1, EGF, and bFGF were added into the bottom chamber, chemotactic migration of the HCC cells occurred in an MMP-independent manner.

 
Regulation of HCC Cell Migration by Profibrotic Growth Factors.
Haptotactic (direct) stimulation of HepG2 and Chang cells with TGF-ß1, EGF, bFGF, or VEGF induced migration into the bottom chamber across the collagen IV/collagen I-coated polycarbonate membrane (Fig. 2A)Citation . Costimulation with TGF-ß1 and EGF, which has been suggested as an optimal stimulus of growth factor-induced epithelial cell migration, had an additive effect (Fig. 2ACitation ; Refs. 10 and 25 ). Chemotactic stimulation (from the bottom chamber) with TGF-ß1, EGF, bFGF, or VEGF attracted HepG2 or Chang cell migration into the bottom compartment within 6 h (Fig. 2B)Citation . Addition of specific neutralizing antibodies inhibited migratory response to each of the respective growth factors. Six h of direct or indirect stimulation with TGF-ß1, EGF, bFGF, or VEGF revealed insignificant differences in proliferation of HepG2 cells or Chang cells, suggesting that increased numbers of these HCC cells in the bottom chamber were exclusively due to enhanced migration (Fig. 2C)Citation .



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  		Fig. 2. A and C, regulation of HCC migration by profibrotic growth factors. Haptotactic migration. Direct stimulation of HepG2 (A) or Chang cells (C) in the top chamber with TGF-ß1 (3 ng/ml) increased migration of HepG2 cells by 2.71-fold and Chang cells by 3.94-fold when compared with the unstimulated control. Stimulation with EGF (10 ng/ml) increased the migration of HepG2 cells by 3.75-fold and Chang cells by 5.68-fold. Stimulation with VEGF (10 ng/ml) increased the migration of HepG2 cells by 3.43-fold and Chang cells by 4.54-fold. Stimulation with bFGF (5 ng/ml) increased the migration of HepG2 cells by 2.39-fold and Chang cells by 3.98-fold. Motility in response to each growth factor was specifically inhibited by blocking antibodies. B and D, chemotactic migration. Addition of TGF-ß1 (3 ng/ml) into the bottom compartment of the Boyden chamber induced migration of HepG2 (B) or Chang cells (D) across the polycarbonate membrane by 3.05-fold or 6.02-fold, respectively. Similarly, addition of EGF induced the migration of HepG2 cell by 4.57-fold and Chang cells by 6.78-fold. The combination of TGF-ß1 and EGF induced the migration of hepG2 cells by 5.36-fold or Chang cells by 8.11-fold. The addition of VEGF induced migration of HepG2 cells by 4.73-fold and Chang cells by 7.10-fold. bFGF induced migration of HepG2 cells by 3.21-fold and Chang cells by 4.67-fold. Blocking antibodies specifically inhibited the migration induced by each growth factor. E, stimulation of HepG2 or Chang cells with TGF-ß1, EGF, TGF-ß1/EGF, VEGF, or bFGF did not induce proliferative activity when compared with unstimulated control cells, as determined by thymidine incorporation assay. #, P < 0.05; **, P < 0.01; ***, P < 0.001 versus unstimulated control.

 
Regulation of HCC Cell Migration by MMPs.
MMPs are considered to facilitate cell invasion by degradation of basement membrane and release of ECM-bound growth factors, creating the stimulus and path for migrating cells (28 , 29) . To investigate the role of MMPs in migration of HCC cells, we first examined the release of MMP-2 and MMP-9 (the major basement membrane degrading MMPs) after direct stimulation of HepG2 or Chang cells with growth factors TGF-ß1, EGF, and bFGF. Stimulation of HepG2 or Chang cells with TGF-ß1, EGF, and bFGF resulted in a significant up-regulation of MMP-2 in the tissue culture supernatants, whereas MMP-9 levels were unaltered (Fig. 3)Citation . Costimulation with TGF-ß1 and EGF, which induced the strongest migratory response, further enhanced MMP-2 levels compared with stimulation with TGF-ß1 or EGF alone, whereas VEGF had no significant effect on either MMP-2 or MMP-9 levels (Fig. 3)Citation . These results suggest that profibrotic growth factors induce release of MMP-2 by HCC cells, potentially contributing to the migration of these cells.



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  		Fig. 3. Regulation of MMPs in HepG2 HCC cells. HepG2 or Chang cells were stimulated with TGF-ß1 (3 ng/ml), EGF (10 ng/ml), a combination of TGF-ß1 and EGF, bFGF (5 ng/ml), or VEGF (5 ng/ml) for 48 h, and the MMPs in the supernatants were assessed by zymography and immunoblot. The panels display representative results of the experiments; the bar graphs summarize densitometric analysis of three independent experiments. Stimulation of HepG2 (A) or Chang (B) cells with TGF-ß1, EGF, or bFGF resulted in a significant increase of MMP-2 activity when compared with the control. Costimulation with TGF-ß1 and EGF had an additive effect on MMP-2 up-regulation. Stimulation with VEGF had no significant effect on MMP-2 activity in the supernatants. None of the growth factors had any significant effect on the up-regulation of MMP-9 activity. B, Immunoblot analysis with supernatants from HepG2 (B) or from Chang (D) cells for MMP-2 and MMP-9 confirmed the increase in MMP-2 production after direct stimulation with TGF-ß1, EGF, TGF-ß1/EGF, or bFGF, whereas VEGF had no significant effect on the expression of MMP-2 and MMP-9. #, P < 0.05; **, P < 0.01.

 
To elucidate further the role of MMPs in HCC cell migration across a fibrotic microenvironment, we subjected fresh HepG2 or Chang cells to conditioned media collected after stimulation of these cells with individual profibrotic growth factors TGF-ß1, EGF, VEGF, and bFGF. Conditioned media of EGF-, TGF-ß1- or bFGF-stimulated HCC cells (with substantially increased levels of MMP-2) increased migration of HepG2 cells and Chang cells significantly. Conditioned media from VEGF-stimulated cells (with insignificant increase of MMP-2) had no significant stimulatory effect on migration of HepG2 or Chang cells (Fig. 4, A and C)Citation , whereas in control experiments, addition of active MMP-2 enzyme in the top chamber was sufficient to induce migration. In these studies, we used supernatant protein at a concentration of 5 µg/ml (equaling a dilution of about 1:1000 of growth factor-containing medium). Supernatant protein contained sufficient amounts of MMPs, whereas the concentration of growth actors was insignificant (about 1 pg/ml). In control experiments, growth factors in the picogram range were insufficient to induce migration. To elucidate further the role of MMP-2 in the conditioned media on HCC cell migration, we used COL-3, a pan-specific small molecule inhibitor of MMP-2/-3/-9 to inhibit MMP activity (27) . Although COL-3 prevented migration that was induced by direct (haptotactic) addition of conditioned media in the supernatant (Fig. 4, A and C)Citation , inhibition of MMP-2 by COL-3 had an insignificant effect on chemotactic migration (conditioned media in the bottom chambers; Fig. 4BCitation ). In summary, these results suggest that direct presence of MMP-2 induces migration of HCC, and inhibition of MMP-2 is a target to prevent haptotactic migration. However, inhibition of MMP-2 does not inhibit chemotaxis, which suggests that chemotactic migration of HCC cells is MMP independent.



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  		Fig. 4. Regulation of HCC cell migration by MMP-2. MMP-2-containing supernatants from HepG2 (A) or Chang (C) cells, stimulated with TGF-ß1 (T-Sup), EGF (E-Sup), VEGF (V-Sup), or bFGF (bF-Sup) were added into the top compartment of the Boyden chamber with the cells at a concentration of 5 µg/ml. T-Sup, E-Sup, and bF-Sup induced migration of HepG2 and Chang cells across the type IV collagen/type I collagen-coated polycarbonate membrane; V-Sup had no significant effect (A and C). The addition of COL-3 (10 ng/ml), an inhibitor of MMP-2 and MMP-9, to the conditioned media stimulated with the growth factors significantly inhibited migration of HepG2 and Chang cells. The decrease was 43.1% with HepG2 cells and 42.6% with Chang cells (T-Sup), 45.6% with HepG2 cells and 44.2% with Chang cells (E-Sup), and 55.7% with HepG2 cells and 40.6% with Chang cells (bF-Sup; A and C, black columns). In the control experiments, addition of MMP-2 in the top chamber induced migration of HepG2 and Chang cells. This effect was inhibited by COL-3. Chemotactic migration of HepG2 (B) and Chang (D) cells was induced by the addition of T-Sup, E-Sup, V-Sup, or bF-Sup into in the bottom chamber. COL-3 had no significant inhibitory effect on chemotactic migration of HepG2 or Chang cells. *, P < 0.05; **, P < 0.01 compared with the control (no COL-3) of each group.

 
Regulation of HCC Cell Migration by Cell-ECM Interactions.
A cycle of attachment and detachment of cells to their underlying ECM via integrins is considered essential for cell migration (16) . In HepG2 cells and Chang cells, substantial levels of {alpha}1ß1 and {alpha}2ß1 are expressed, suggesting that these two collagen-binding integrins are common to these two HCC cells (Fig. 5)Citation . To evaluate the role of {alpha}1ß1 and {alpha}2ß1 integrins in migration of HCC cells, we used {alpha}1ß1 and {alpha}2ß1 integrin-blocking antibodies. Blocking of {alpha}1, {alpha}2, and ß1 integrins significantly inhibited both chemotactic (Fig. 6, A and C)Citation and haptotactic (Fig. 6, B and D)Citation migration that was induced by TGF-ß1, EGF, or bFGF. {alpha}1 and {alpha}2 Integrins associate with ß1 integrin subunit, and blocking of ß1 integrin also resulted in additional inhibition of migration (Fig. 6)Citation . Blocking of {alpha}3 integrin in this setting caused a mild (statistically insignificant) decrease in the haptotactic and chemotactic migration of both HepG2 and Chang cells across the collagen IV/collagen I-coated membrane (Fig. 6, A–D)Citation . In summary, these findings demonstrate that migration of HepG2 and Chang cells is dependent on pathogenic cell-ECM interactions via {alpha}1ß1 and {alpha}2ß1 integrins.



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  		Fig. 5. Integrin expression on HepG2 and Chang cells. HepgG2 and Chang cells were incubated with antibodies to {alpha}1, {alpha}2, {alpha}3, or ß1 integrins. Integrin expression is displayed by a shift in mean fluorescent intensity compared with no primary antibody incubation using fluorescence-activated cell sorting analysis. The percentages of positive cells for expression of integrin subunits, when compared with control incubations, are indicated on each plot. A, HepG2 cells abundantly express to {alpha}1, {alpha}2, and ß1, whereas {alpha}3 staining was almost insignificant. B, Chang cells expressed {alpha}1, {alpha}2, {alpha}3, and ß1 integrins.

 


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  		Fig. 6. Regulation of HCC cell migration by integrins. A and C, haptotactic migration. Migration of HepG2 (A) and Chang (C) cells was induced by addition of 3 ng/ml TGF-ß1, 10 ng/ml EGF, or 5 ng/ml bFGF into the top chamber, together with the blocking antibodies to {alpha}1, {alpha}2, {alpha}3, and ß1 integrins (5 µg/ml). Migration induced by each growth factor, TGF-ß1, EGF, or bFGF, was significantly inhibited with the {alpha}1, {alpha}2, and ß1 integrin antibodies, but not with the {alpha}3 antibody. B and D, chemotactic migration. Chemotactic migration of HepG2 (B) and Chang (D) cells was induced by addition of TGF-ß1, EGF, or bFGF into the bottom chamber. The blocking antibodies to {alpha}1, {alpha}2, {alpha}3, and ß1 integrins were added into the top chamber. Blocking {alpha}1, {alpha}2, and ß1 integrin antibodies significantly inhibited chemotactic migration induced by TGF-ß1, EGF, or bFGF. However, blocking {alpha}3 integrin antibody had only insignificant inhibitory effect. *, P < 0.05; **, P < 0.01.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Invasion and metastasis are important determinants in the progression of HCC (7) . Motility of cancer cells is dependent on their interactions with their microenvironment (14) . Stimulation by cytokines and growth factors, cleavage of basement membrane boundaries, and cell attachment to fibrillar ECM are considered major regulators of migration (14) . HCC is almost inevitably associated with a cirrhotic microenvironment, which is characterized by abundance of profibrotic growth factors and an altered ECM composition (3 , 9 , 30, 31, 32, 33, 34) .

Here, we demonstrate that profibrotic growth factors TGF-ß1, EGF, and bFGF induce haptotactic as well as chemotactic migration of HepG2 (minimally invasive HCC cells) and Chang (highly invasive HCC cells) cells. Similar migratory response of these two cell lines, which possess different migratory capacities per se, suggests that the fibrotic microenvironment accounts for regulation of migratory behavior, independent of a potential fibrotic HCC cell phenotype. Our results support previous observations, which demonstrated that HCC cells possess limited intrinsic invasive capacity in absence of growth factors, whereas TGF-ß1 induces migratory capacity (20 , 21) . These growth factors are known to be released by stromal cells in the cirrhotic liver, suggesting that antifibrotic therapies that target stromal cells can provide additional benefit by decreasing levels of promigratory growth factors (9) . It might be possible that the other growth factors could compensate for the effect of blocking a single growth factor (35 , 36) . This suggests that targeting a single promigratory growth factor may not be of significant therapeutic benefit, and our experimental protocol addressed this issue. Thus, we performed additional studies to identify common downstream mediators of growth factor-induced HCC cell migration.

MMPs are considered to play an important role in the invasion of cancer cells by degradation of basement membranes (18 , 37) . We demonstrate that haptotactic migration of HepG2 and Chang cells is dependent on MMP-2, which is released by stimulated HepG2 or Chang cells (27) . In our experiments, chemotactic migration of HCC cells was MMP independent. Chemotactic induction of migration observed in the present study could represent events induced by concentration gradients and possible induction of cellular polarity in the top chamber, with the tips of the cells nearest the matrix/TGF-ß1 protein expressing focal membrane-associated MMPs and promigratory integrins, resulting in amoeboid-like migration. Such amoeboid-like migration would result in the cells squeezing through the matrix pores without degradation. In contrast, haptotactic migration leads to direct receptor-mediated growth factor action on cells to express MMPs and integrins evenly, which lead to migration of cells dependent on direct degradation of matrix molecules. Our results are supported by observations that suggest that MMPs are conducive for migration across ECM, but are not an absolute requirement, because malignant cells can invade ECM in an integrin-dependent manner in the absence of MMPs (24 , 38) . These results suggest that the therapeutic potential of MMP inhibition of HCC might be limited.

Altered cell-ECM interactions are considered important in the invasion and metastasis of tumor cells (8 , 14) . In the cirrhotic liver, type I collagen and type IV collagen are the major ECM constituents, whereas basement membrane proteins with cell-binding capacity, such as laminins, are replaced by fibrillar collagens (30) . In cell motility assays using basement membrane constituents, integrins {alpha}3ß1, {alpha}6ß1, {alpha}vß1, and {alpha}vß5 were identified as important mediators of HCC cell invasion (20, 21, 22, 23 , 39) . However, although these studies were important in elucidating the role of resident basement membrane constituents for migration, our study takes into account the ECM switch to a matrix rich in fibrillar ECM during liver fibrosis associated with HCC.

Our results demonstrate that inhibition of {alpha}1, {alpha}2, and ß1 integrins significantly inhibits chemotactic and haptotactic migration induced by individual growth factors TGF-ß1, EGF, or bFGF. Furthermore, recent studies suggest that inhibition of {alpha}1ß1 integrin also potentially inhibits fibrogenesis and thus could potentially further delay invasion and progression of HCC (40) . In our experiments, blocking of {alpha}3 integrin had only a mild inhibitory effect on the migration of HCC cells. In this regard, whereas {alpha}3ß1 integrin is expressed by some HCC cells, it is mainly considered to be a laminin receptor. In this study, we used type I collagen and type IV collagen to coat the two sides of the membrane to mimic the fibrotic microenvironment and not laminin (which is replaced in fibrosis).

The expression levels of many different integrins are up-regulated during cancer progression. Whereas on normal hepatocytes, {alpha}5ß1 and {alpha}6ß1 are the major integrins (41, 42, 43, 44) , carcinogenesis of hepatocytes is associated with a switch in integrin expression, and integrins {alpha}1ß1, {alpha}2ß1 and {alpha}3ß1 are abundantly expressed (20 , 45 , 46) . This switch of integrin expression is associated with acquisition of migratory capacity (primary human hepatocytes are immobile), suggesting an important role of these integrins in HCC cell motility. Furthermore, several growth factors, which are abundantly expressed in the cirrhotic tumor microenvironment, are considered potent stimulators of integrin expression (47 , 48) .

Collectively, our findings suggest that growth factor-induced migration of HepG2 and Chang cells can be inhibited by blocking the function of {alpha}1ß1 and {alpha}2ß1 integrins. Although MMP-2 inhibition could result in some benefit, integrin-based therapy might be more effective in treating metastatic HCC.


   ACKNOWLEDGMENTS
 
We thank Collagenex, Inc. for their gift of COL-3.


   FOOTNOTES
 
Grant support: The Espinosa Fibrosis Fund (C. Y.), NIH Grants DK62987 and DK55001, The Center for Matrix Biology at the Beth Israel Deaconess Medical Center (C. Y.), Deutsche Forschungsgemeinschaft Grant DFG ZE 523 1/1 (M. Z.), Emil Aaltonen Foundation (P. N.), The Cancer Society of Finland (P. N.), The Stop and Shop Pediatric Brain Tumor Foundation (J. L., M. Z.), and The National Research Service Award 1 F32 CA101436-01 from the National Cancer Institute (J. L.).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Requests for reprints: Dr. Raghu Kalluri, Harvard Medical School, Center for Matrix Biology, Beth Israel Deaconess Medical Center, Department of Medicine, Dana 514, 330 Brookline Avenue, Boston, Massachusetts 02215. Phone: (617) 667-0445; Fax: (617) 975-5663; E-mail: rkalluri{at}bidmc.harvard.edu

1 The abbreviations used are: HCC, hepatocellular carcinoma; ECM, extracellular matrix; TGF-ß1, transforming growth factor ß; EGF, epidermal growth factor; bFGF, basic fibroblast growth factor; MMP, matrix metalloproteinase; VEGF, vascular endothelial growth factor; EMEM, Eagle’s minimum essential medium; FBS, fetal bovine serum. Back

Received 2/ 6/03. Revised 9/16/03. Accepted 10/ 2/03.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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