Invasive breast cancer induces laminin-332 upregulation and integrin β4 neoexpression in myofibroblasts to confer an anoikis-resistant phenotype during tissue remodeling
© Cho et al.; licensee BioMed Central Ltd. 2012
Received: 1 February 2012
Accepted: 6 June 2012
Published: 6 June 2012
Although development of anoikis-resistant myofibroblasts during tissue remodeling is known to be associated with tumor invasion, the mechanism by which myofibroblasts become resistant to anoikis is unknown. We previously demonstrated laminin-332 upregulation in the fibrosis around invasive ductal carcinoma (IDC). Because laminin-332 promotes cell survival through binding to integrins, we hypothesized that invasive breast cancer cells confer an anoikis-resistant phenotype on myofibroblasts by upregulating laminin-332 expression during tissue remodeling. Here, we demonstrate that invasive breast cancer cells induce laminin-332 upregulation and integrin β4 neoexpression in myofibroblasts to confer an anoikis-resistant phenotype.
Three types of fibroblasts were isolated from the tumor burden, the fibrosis, and normal tissue of patients with early stage IDC (less than 10 mm diameter), designated cancer-associated fibroblasts (CAFs), interface fibroblasts (InFs), and normal breast fibroblasts (NBFs), respectively. To investigate direct and indirect crosstalk with tumor cells, fibroblasts were co-cultured with invasive MDA-MB-231 or noninvasive MCF7 cells or in conditioned medium. Anoikis resistance of fibroblasts was measured by cell viability and caspase-3 activity after incubation on poly-HEMA coated plates for 72 hours. Involvement of laminin-332/integrin α3β1 or α6β4 signaling in anoikis resistance was confirmed by treatment with purified laminin-332 or blocking antibodies against laminin-332, integrin β1, or integrin β4.
MDA-MB-231 cells induced laminin-332 upregulation and integrin β4 neoexpression in fibroblasts, leading to anoikis resistance. InFs showed a higher endogenous level of laminin-332 than did CAFs and NBFs. After stimulation with MDA-MB-231-conditioned medium, laminin-332 expression of InFs was dramatically increased and maintained under anoikis conditions. Laminin-332 upregulation was also observed in CAFs and NBFs, but at a lower level than in InFs. Laminin-332 induced Akt (Ser473) phosphorylation by binding to integrin α3β1. Integrin β4 neoexpression induced laminin-332-independent Rac1 activation and promoted anoikis resistance in fibroblasts approximately twofold more effectively than did laminin-332, regardless of the type of fibroblast. In addition, integrin β4 expression suppressed fibroblast aggregation in conditions of anoikis.
Invasive breast cancer cells confer an anoikis-resistant phenotype on myofibroblasts during tissue remodeling by inducing laminin-332 upregulation and integrin β4 neoexpression. Interface fibroblasts appear to be the primary myofibroblasts that interact with invasive tumor cells during tissue remodeling.
A fundamental component of tumor invasion is stromal tissue remodeling, which involves proteolytic degradation of the extracellular matrix (ECM) and results in anoikis (a form of caspase-dependent apoptosis that is caused by loss of integrin binding of stromal cells) . As a component of stroma, myofibroblasts are also exposed to anoikis during tissue remodeling. However, many studies have reported prolonged survival of myofibroblasts during tissue remodeling in patients with fibrotic diseases [2–5]. Fibrosis is considered an indicator of tissue remodeling  and is commonly formed around invasive types of tumors [7–9]. Considering that myofibroblasts are key regulators of tissue remodeling  and a major source of ECM production , which drives tumor progression, the development of anoikis-resistant myofibroblasts may be an essential event during stromal tissue remodeling before tumor invasion.
Abnormal and excessive ECM deposition not only is a phenotype of fibrosis but also is associated with cell-survival signaling mediated by integrin receptors during tumor invasion and tissue remodeling . Therefore, altered molecular expression in fibrosis may provide a clue to how myofibroblasts acquire an anoikis-resistant phenotype during tissue remodeling. Previously, we observed aberrant laminin-332 upregulation in the fibrosis of invasive ductal carcinoma (IDC) compared with autologous tumor burden and distal normal tissue, whereas such laminin-332 upregulation was not found in the noninvasive counterpart, ductal carcinoma in situ (DCIS) . Contrary to our finding, laminin-332 expression was previously reported to be downregulated in breast cancer . Laminin-332, a large multidomain molecule involved in cell adhesion and matrix assembly, plays an important role in cell migration and survival by activating many signal mediators through binding to integrin α3β1 or α6β4 [15–18]. The role of laminin-332 in cell survival or anoikis resistance has been studied mostly in epithelial tumor cells, although some data exist on laminin-332-dependent survival of keratinocytes during wound healing [19, 20]. One group demonstrated that the matricellular protein thrombospondin1 (TSP1) induces anoikis resistance in mouse embryonic fibroblasts in vitro . This work provided convincing evidence that an ECM protein can induce anoikis resistance in fibroblasts and suggested that laminin-332 may fulfill a similar function. However, because the fibroblasts used in previous studies were not derived from tumor stroma, the role of anoikis resistance of myofibroblasts in the context of tumor invasion remains undetermined.
In this study, we hypothesized that invasive breast cancer cells induce laminin-332 upregulation in myofibroblasts undergoing tissue remodeling, which leads to anoikis resistance through laminin-332 autocrine signaling. To test our hypothesis, we used three types of fibroblasts isolated from IDC tissue and co-cultured them with breast cancer cells to investigate tumor-stroma crosstalk. Our results provide evidence that anoikis-resistant myofibroblasts develop during tissue remodeling of invasive breast cancer as a result of abnormal upregulation of ECM proteins and/or their receptors.
Materials and methods
Isolation of primary fibroblasts
Human breast tumors and autologous normal breast tissues were obtained from three IDC patients undergoing surgery at Severance Hospital of the Yonsei University Health System, Korea. The protocol for the study was approved by Severance Hospital Ethics Committee (4-2008-0380). All participants signed written informed consent forms detailing tissue use for comprehensive experiments on breast cancer. Based on the zonal concept proposed in our previous study, primary fibroblasts were isolated from tissue in the tumor zone (TZ, tumor burden), interface zone (IF, fibrosis), and normal zone (distal normal tissue), as described previously .
Direct and indirect co-culture
For direct co-culture, primary fibroblasts were cultured with MCF7 or MDA-MB-231 breast cancer cells (Korean Cell Line Bank, Seoul, Korea), as described previously . MDA-MB-231 and MCF7 cells were selected as representative invasive and noninvasive types of breast cancer, respectively. Adherent fibroblasts were stained with 5 μM Cell Tracker Green CMFDA (5-chloromethylfluorescein diacetate; Invitrogen, Eugene, OR, USA) by incubation at 37°C for 45 minutes. The dye solution was then replaced with fresh prewarmed medium, and the cells were incubated at 37°C for another 2 hours. After washing them twice with PBS, unstained cancer cells were plated onto a layer of CMFDA-stained fibroblasts. The co-cultures were incubated in reduced serum media (RSM) composed of DMEM/F12, 1% FBS, 100 IU/ml penicillin, and 100 μg/ml streptomycin for 1 week. CMFDA-stained fibroblasts and unstained breast cancer cells were sorted with flow cytometry and used for subsequent experiments. For indirect co-culture, conditioned medium (CM) was prepared by culturing MCF7 or MDA-MB-231 breast cancer cells at 80% confluency in RSM for 1 day. The CM was filtered through a 0.22-μm filter (Millipore, Billerica, MA, USA) and diluted 1:1 with fresh RSM before addition to fibroblast cultures, which were incubated for a further 3 days.
Transfection of fibroblasts with integrin β4
For transient transfection with integrin β4, fibroblasts (5 × 105 cells/well) were plated on a six-well plate and transfected with 3 μg pRK6 b4 plasmid DNA (Addgene plasmid 16037; Addgene, Cambridge, MA, USA; kindly donated by Dr. FG Giancotti, Cellular Biochemistry and Biophysics Program, Memorial Sloan-Kettering Cancer Center, New York, NY, USA) , and 3 μl MATRA (Promokine, Heidelberg, Germany) in a total volume of 200 μl Opti-MEM on a Universal Magnet Plate (Promokine) for 15 minutes. Controls were transfected with 3 μg pRK5.
Fibroblasts (5 × 105 cells/well in 1 ml) were added to either poly-HEMA- or serum-coated wells and incubated at 37°C in 5% CO2 with RSM, MCF-7 CM, or MDA-MB-231 CM for 6, 24, 48, or 72 hour. Maintenance of suspended fibroblasts in poly-HEMA-coated wells (anoikis conditions) was monitored by phase microscopy. Cell viability was measured by addition of 100 µl CCK-8 (Dojindo Molecular Technologies, Inc., Rockville, MD, USA) to each well. After incubation at 37°C for 2 hours, fluorescence was measured with a plate reader (excitation, 530 nm/emission, 580 nm). Fibroblasts from three IDC patients were assayed in triplicate in at least three separate experiments. For function-blocking assays, antibodies against integrin β1, β4, or laminin-332 (Millipore) were added to InF cells (which have high expression of laminin-332) with MDA-MB-231 CM and incubated at 37°C in a humidified 5% CO2 incubator for 24 hours. CAF cells (which express low levels of laminin-332) were treated with purified laminin-332 (Abcam, Cambridge, England) in RSM for 24 hours.
Caspase-3 activity assay
Caspase-3 activity in fibroblast lysates was measured by using a caspase-3 activity kit (Becton-Dickinson-Pharmingen, San Jose, CA, USA). Fibroblasts (5 × 105 cells) were plated in poly-HEMA-coated-wells with RSM, MCF7 CM, or MDA-MB-231 CM and treated with blocking antibodies or purified laminin-332 for 24 hours. Fibroblast lysates were incubated in a 96-well plate in HEPES buffer with 5 µl caspase-3 fluorogenic substrate (Ac-DEVD-AMC) for 1 hour at 37°C in the dark. Fluorescence was measured with a plate reader (excitation, 360 nm/emission, 460 nm).
Rac1 pull-down assay
Suspended fibroblasts were harvested by centrifugation and lysed in 1× lysis buffer. Rac/cdc42 assay reagent (10 µl; 10 µg) was added to 0.5 ml cell lysate, and the reaction mixture was incubated by rocking gently at 4°C for 1 hour. Agarose beads were collected by pulsing for 5 seconds in a microcentrifuge at 14,000 g, and the supernatant was discarded. The pelleted beads were washed 3 times with 0.5 ml Mg2+ Lysis/Wash Buffer (MLB), resuspended in 40 µl 5× Laemmli reducing sample buffer, boiled for 5 minutes, separated by polyacrylamide gel electrophoresis (PAGE), and then detected with immunoblot.
Suspended cells were collected by centrifugation, lysed in 100 μl Protein Extraction Solution (Intron Biotech, Seongnan-si, Korea), homogenized with a 30-gauge needle, incubated for 30minutes at 4°C, and then purified by centrifugation at 13,000 rpm. After quantifying proteins in the extracts by using the Bradford method, 20 μg protein was electrophoresed on 10% Tris/glycine gels (Invitrogen, Carlsbad, CA, USA), transferred to polyvinylidine fluoride (PVDF) membranes (Millipore), and probed with primary antibodies against laminin γ2, integrin α3, integrin α6, integrin β1, integrin β4, Akt, phospho(Ser473)-Akt, E-cadherin, β-catenin, GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA, USA), or Rac1 (Millipore). Primary antibodies were detected with horseradish peroxidase (HRP)-conjugated secondary antibodies (Invitrogen), and visualized by using enhanced chemiluminescence reagents (Santa Cruz Biotechnology).
Statistical significance (indicated by asterisks) was determined by using the t test and ANOVA. Results were considered to be significant at P < 0.05. All statistical analyses were performed by using SPSS version 11.5 for Windows statistical software (SPSS Inc.).
Invasive breast cancer cells induce upregulation of laminin-332 expression in stromal fibroblasts
Integrin β4 neoexpression in fibroblasts is induced through direct contact with invasive breast cancer cells
Anoikis resistance of fibroblasts is conferred by binding of laminin-332 to integrin α3β1 and by neoexpression of integrin β4
To confirm that fibroblast viability was associated with binding of laminin-332 to integrins α3β1 and/or α6β4, InFs and InFs/β4 (expressing integrin β4) were first stimulated with MDA-MB-231 CM to induce laminin-332 upregulation and then treated with blocking antibodies against laminin-332, integrin β1, or integrin β4. The viability of both InF and InFs/β4 was inhibited by blocking integrin β1 and laminin-332, whereas blocking integrin β4 had little effect (Figure 3E).
To show that the viability of fibroblasts depends on laminin-332 expression, CAFs (Ln-332-low) were treated with 10 μM purified laminin-332, resulting in increased viability. The viability of CAFs/β4 was strongly maintained, independent of laminin-332 treatment (Figure 3F).
Laminin-332 upregulation and integrin β4 neoexpression suppress caspase-3 activity in fibroblasts
Similar to these results, induction of integrin β4 also reduced caspase-3 activity (Figure 4B). To show further that the caspase-3 activity of fibroblasts was decreased by laminin-332 upregulation, CAFs (Ln-332-low) and CAFs/β4 were treated with 10 μM purified laminin-332, and the resulting caspase-3 activities were measured. Treatment with laminin-332 significantly decreased caspase-3 activity in both CAFs and CAFs/β4 (Figure 4C).
Enhancement of anoikis resistance by integrin β4 neoexpression is mediated by Rac1 activation
Integrin β4 neoexpression suppresses aggregation of fibroblasts under anoikis conditions
In the present work, we hypothesized that myofibroblasts gain an anoikis-resistant phenotype during tissue remodeling through laminin-332 upregulation and integrin β4 neoexpression induced by interaction with invasive breast cancer cells. To prove our hypothesis, we isolated three types of fibroblasts, CAFs from the tumor zone (the epicenter of tumor burden), InFs from the interface zone (fibrosis), and NBFs from the normal zone (distal normal tissue) of IDC, and co-cultured them with tumor cells to investigate the tumor-stroma interaction. InFs, the myofibroblasts undergoing tissue remodeling, showed a higher resistance to anoikis than did CAFs and NBFs as a result of their high endogenous expression level of laminin-332 and its dramatic upregulation by conditioned medium from invasive MDA-MB-231 cancer cells. In addition, integrin β4 neoexpression was induced in fibroblasts by cell-to-cell contact with MDA-MB-231 cells regardless of the type of fibroblast, and expression of integrin β4 increased anoikis resistance to a level approximately twofold higher than that induced by laminin-332 upregulation. From these results, we conclude that invasive breast cancer cells confer an anoikis-resistant phenotype on myofibroblasts during tissue remodeling by inducing laminin-332 upregulation and integrin β4 neoexpression, and that InFs may be the primary myofibroblasts involved in tissue remodeling during invasion of breast cancer cells.
This may be the first study to describe how myofibroblasts acquire an anoikis-resistant phenotype during tissue remodeling by using a model of invasive breast cancer. Previously, one group reported that transforming growth factor β1 (TGF-β1) confers an anoikis-resistant phenotype on normal primary human fetal lung fibroblasts , and another group showed that the matricellular protein TSP1 induces anoikis resistance in mouse embryonic fibroblasts . However, the fibroblasts used in these studies were not derived from tumor stroma, and we believed that it was necessary to investigate the development of anoikis-resistant myofibroblasts in the context of tumor progression. Therefore, in this study, we used IDC to examine the development of anoikis-resistant myofibroblasts during tissue remodeling.
We chose to study IDC for two reasons: the first was the dense fibrosis around the tumor burden, which is a phenotype of vigorous tissue remodeling [7–9], and the second was aberrant upregulation of laminin-332 in the fibrosis (in contrast, laminin-332 upregulation was not detected in its counterpart, DCIS) . Because laminin-332 is widely known to promote cell survival, the anoikis resistance of myofibroblasts is likely mediated by laminin-332-dependent signaling. To test this assumption, we first investigated laminin-322 expression in CAFs, InFs, and NBFs and found that the expression pattern in the fibroblasts was the same as that in situ (Figure 1A). Based on α-SMA expression and their location in the tissue, CAFs and InFs are presumed to be the myofibroblasts that interact with invasive tumor cells during tissue remodeling.
Within the interface zone (IZ) adjacent to the tumor zone (TZ), invasive breast cancer cells may interact with InFs via cell-to-cell contact at the border between the TZ and IZ or with diffusible factors away from the border and within the IZ. It is therefore possible that the expression of laminin-332 or its receptors, integrin α3β1 and α6β4, is affected differently according to the type of interaction. To test this notion, fibroblasts were co-cultured with tumor cells to simulate cell-to-cell contact or were stimulated with the CM from tumor cells to test the role of diffusible factors. In our previous study, laminin-332 upregulation in the IZ was shown to be associated with tumor invasiveness . Thus, MDA-MB-231 and MCF7 were used as invasive and noninvasive breast cancer cell types, respectively. After treatment with MDA-MB-231 CM, but not MCF7 CM, laminin-γ2, a subunit used to identify laminin-332, was upregulated in InFs, and this upregulation was maintained under anoikis conditions. Expression of laminin-γ2 in CAFs and NBFs was also promoted by MDA-MB-231 CM, but to a lesser extent than in InFs (Figure 1C). The degree of laminin-γ2 induction by diffusible factors produced by MDA-MB-231 seemed to depend on the endogenous capacity of fibroblasts to express laminin-γ2. According to previous reports, TGF-β is a major soluble factor produced by MDA-MB-231 [26–28]. Therefore, we examined whether laminin-γ2 upregulation by MDA-MB-231 CM is induced by TGF-β and found that the TGF-β inhibitor LY2157299 effectively suppressed laminin-γ2 expression (see Additional file 1, Figure S1). Based on this result, we suggest that expression of laminin-322 is at least partially induced by TGF-β secreted by invasive breast cancer cells. However, further evaluation is required because we did not test other cytokines that are highly secreted from invasive breast cancer cells. In contrast to laminin-332 upregulation by MDA-MB-231 CM, integrins α3, α6, and β1 were highly expressed in fibroblasts and not affected by either diffusible factors or direct contact with MDA-MB-231 cells (Figure 2, and see Additional file 1, Figure S3). Integrin β4 was not endogenously expressed in fibroblasts, but its expression was newly induced by direct contact with MDA-MB-231 cells. Almost no difference was found in the induction of anoikis resistance by integrin β4 neoexpression in the different types of fibroblasts.
When in direct contact with invasive breast cancer cells, the anoikis resistance of InFs involved both integrin α3β1 and integrin α6β4 as a result of integrin β4 neoexpression. Although both integrins are known receptors for laminin-332, their signaling pathways, signal duration, and dependency on laminin-332 are different. In the case of integrin α3β1, signaling was dependent on laminin-332 binding and mediated by Akt (Ser473) phosphorylation (Figure 5A). In contrast, integrin α6β4-mediated signaling was independent of laminin-332 and mediated through Rac1 activation (Figure 5B). Anoikis resistance induced by integrin α6β4 was approximately twofold higher than that induced by integrin α3β1 (Figure 3D). This is supported by a report that Rac1 activation through integrin α6β4 produces a long-lasting survival signal . Furthermore, the long cytoplasmic tail of integrin β4 can be phosphorylated by other factors such as c-Met, independent of laminin-332 . In addition to anoikis resistance, integrin β4 neoexpression suppressed fibroblast aggregation under anoikis conditions, possibly as a result of downregulation of β-catenin (Figure 6D). Many cells overcome anoikis through synoikis, the formation of cell clusters via E-cadherin-mediated interaction between cells. The expression of E-cadherin on the cell surface is known to be regulated by β-catenin ; therefore, decreased expression of β-catenin by integrin β4 neoexpression may cause defective surface expression of E-cadherin, which in turn suppresses fibroblast aggregation. Considering a recent report that myofibroblasts in the primary tumor site metastasize to the lung along with tumor cells , in the context of tumor metastasis, single cells or a small aggregate of myofibroblasts may be more likely to undergo distant migration with invasive tumor cells than might a large cluster.
Second, direct contact between invasive breast cancer cells and myofibroblasts induces integrin β4 neoexpression in the fibroblasts. Consequently, myofibroblasts express both integrin α3β1 and integrin α6β4 as receptors for laminin-332. In this case, cell-survival signals are mediated by Rac1 activation and Akt (Ser473) phosphorylation. Akt (Ser473) phosphorylation requires binding of laminin-332 to integrin α3β1, whereas Rac1 activation by integrin α6β4 is not dependent on laminin-332. Furthermore, integrin β4 neoexpression suppresses fibroblast aggregation under anoikis conditions, possibly through downregulation of β-catenin.
Invasive breast cancer cells confer an anoikis-resistant phenotype on myofibroblasts during tissue remodeling by inducing laminin-332 upregulation and integrin β4 neoexpression. Our findings indicate that interface fibroblasts may be the primary myofibroblasts involved in tissue remodeling during breast cancer invasion.
cancer-associated fibroblast (fibroblasts isolated from the TZ)
ductal carcinoma in situ
invasive ductal carcinoma
interface fibroblast (fibroblasts isolated from the IZ)
interface zone (tumor-free fibrotic zone adjacent to tumor burden)
normal breast fibroblast (fibroblasts isolated from the NZ)
normal zone (normal tissue neighboring the IZ)
tumor zone (tumor burden of IDC).
This study was supported by the Korea Healthcare Technology R&D Project, Ministry for Health, Welfare and Family Affairs, Republic of Korea (CNH A101264) and by a faculty research grant of Yonsei University College of Medicine for 2011(6-2011-0106; CNH).
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