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. 2012 Jun 6;14(3):R88.
doi: 10.1186/bcr3203.

Invasive breast cancer induces laminin-332 upregulation and integrin β4 neoexpression in myofibroblasts to confer an anoikis-resistant phenotype during tissue remodeling

Baek Gil Kim  1 , Ming-Qing Gao, Yoon Pyo Choi, Suki Kang, Haeng Ran Park, Kyu Sub Kang, Nam Hoon Cho
Affiliations

Invasive breast cancer induces laminin-332 upregulation and integrin β4 neoexpression in myofibroblasts to confer an anoikis-resistant phenotype during tissue remodeling

Baek Gil Kim et al. Breast Cancer Res. .

Abstract

Introduction: 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.

Methods: 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.

Results: 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.

Conclusion: 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.

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Figures

Figure 1
Figure 1
Laminin-332 expression in stromal fibroblasts is upregulated by invasive breast cancer cells. (A) Isolation of stromal fibroblasts from invasive ductal carcinoma (IDC). Based on the zonal concept introduced in our previous study, fibroblasts were isolated from early-stage IDC tissue (less than 10 mm diameter) by enzyme digestion after confirmation of laminin-γ2 expression. (B) Expression of laminin-γ2 and α-SMA in fibroblasts of IDC. (C) Stimulation of laminin-γ2 expression in fibroblasts by CM from cancer cells. Fibroblasts were incubated with RSM, MCF7 CM, or MDA-MB-231 CM for 24 hours. (D) Duration of laminin-γ2 expression in interface fibroblasts (InFs) under anoikis conditions. InFs (5 × 105 cell/well) were seeded onto poly-HEMA coated six-well plates with RSM or MDA-MB-231 CM for 72 hours. Suspended cells were collected at 6, 24, 48, and 72 hours, as indicated.
Figure 2
Figure 2
Integrin β4 neoexpression is induced in fibroblasts through direct contact with invasive breast cancer cells. (A) Endogenous expression of integrin β1 and β4 in fibroblasts of IDC. Fibroblasts were prestained with CMFDA and cultured in RSM, MCF7 CM, or MDA-MB-231 CM for 72 hours before staining with PE-conjugated anti-integrin β1 or β4 antibodies. (B) Integrin β1 expression and (C) Integrin β4 expression in fibroblasts cocultured with MCF7 or MDA-MB-231 cells. Confluent fibroblasts on 100-mm dishes were stained with 5 μM CMFDA. MCF7 or MDA-MB-231 cells (5 × 105) were added to the stained fibroblasts and cocultured for 1 week. Mixed cells were collected and stained with PE-conjugated anti-integrin β1 or β4 antibodies. Integrin β1 or β4 expression in cocultured fibroblasts was confirmed by immunoblot analysis after sorting.
Figure 3
Figure 3
The anoikis resistance of fibroblasts is mediated by binding of laminin-332 to integrin α3β1 and/or integrin β4 neoexpression. Viability of wild-type and integrin β4-expressing (A) InFs, (B) CAFs, and (C) NBFs in the absence or presence of stimulation from cancer cells under anoikis conditions. The figures show fibroblast viability in RSM (open square), MCF7 CM (open diamond), and MDA-MB-231 CM (open circle) in the absence of integrin β4 expression, and viability in RSM (black square), MCF7 CM (black diamond), and MDA-MB-231 CM (black circle) in the presence of integrin β4 expression. (D) Direct comparison of fibroblast viabilities. The viability of wild-type and integrin β4-expressing fibroblasts was compared at 72 hours. Black bar, CAFs; gray bar, InFs; white bar, NBFs; gray box, integrin β4-expressing fibroblasts. (E) Inhibition of the interaction between laminin-332 and integrin. InF and InF/β4 cells were treated with isotype (black bar) and blocking antibodies against integrin β1 (dark gray bar), β4 (light gray bar), and laminin-332 (white bar) for 24 hours under anoikis conditions. (F) Increased viability of CAFs in response to treatment with laminin-332. CAF and CAF/β4 cells were treated with purified laminin-332 (diluted to 10 μM with RSM) in poly-HEMA-treated 96-well plates for 24 hours. Black bar, untreated CAFs; white bar, laminin-322-treated CAFs. Results are expressed as mean ± SD. *P < 0.05; **P < 0.03 versus RSM in poly-HEMA-coated wells. Results are averages of three separate experiments.
Figure 4
Figure 4
Laminin-332 upregulation and integrin β4 neoexpression suppressed caspase-3 activity in fibroblasts. (A) Caspase-3 activity of fibroblasts under anoikis conditions in the absence or presence of integrin β4 and/or stimulation with CM from cancer cells. Black bar, CAF; gray bar, InF; white bar, NBF; gray box, integrin β4-expressing fibroblasts. (B) Caspase-3 activity according to inhibition of integrin β1 (dark-gray bar), β4 (light-gray bar), or laminin-332 (white bar). The black bar shows the isotype control. (C) Effect of laminim-322 treatment on caspase-3 activity in CAFs. Black bar, untreated CAFs; white bar, laminin-322-treated CAFs. Results are expressed as mean ± SD. *P < 0.05; **P < 0.03 versus RSM in poly-HEMA-coated wells. Results are averages of three separate experiments.
Figure 5
Figure 5
Integrin β4 neoexpression induces Rac1 activation. Akt phosphorylation and Rac1 activation is shown in (A) wild-type and (B) integrin β4-expressing fibroblasts. Wild-type and integrin β4-expressing fibroblasts were incubated in RSM, MCF7 CM, or MDA-MB-231 CM under anoikis conditions for 24 hours. Suspended fibroblasts were harvested for Rac1 pull-down assays.
Figure 6
Figure 6
Integrin β4 neoexpression suppresses fibroblast aggregation under anoikis conditions. Formation of fibroblast aggregates under anoikis conditions in (A) the absence or (B) the presence of integrin β4. Fibroblasts (5 × 105) were plated with RSM, MCF7 CM, or MDA-MB-231 CM on poly-HEMA-coated six-well plates and incubated for 24 hours. The formation of cell aggregates was observed with phase microscopy. (C) The scattering of integrin β4-expressing fibroblasts under anoikis conditions was not affected by blocking the laminin-332/integrin interaction. Fibroblasts (2.5 × 104) were plated with RSM, MCF7 CM, or MDA-MB-231 CM on poly-HEMA-coated 96-well plates with blocking antibodies against integrin β1 or β4 (diluted 1:100 with RSM) or MDA-MB-231 CM for 24 hours. (D) Decreased expression of β-catenin may cause reduced fibroblast aggregation. Suspended cells were collected, lysed, and subjected to Western blot analysis of β-catenin expression.
Figure 7
Figure 7
Two possible mechanisms by which myofibroblasts acquire anoikis resistance via integrin receptors for laminin-332 during tissue remodeling. Direct and indirect interactions may occur between invasive tumor cells and myofibroblasts in the interface zone of invasive ductal carcinoma. Myofibroblasts within the interface zone could be stimulated to express laminin-332 by diffusible factors from invasive breast cancer cells. Binding of laminin-332 to integrin α3β1 on the myofibroblasts turns on a cell-survival signaling pathway mediated by Akt phosphorylation. In this case, myofibroblasts may simultaneously use synoikis to overcome anoikis. In addition, in the border between the tumor zone and the interface zone, invasive breast cancer cells can directly interact with myofibroblasts, leading to integrin β4 neoexpression. Once integrin β4 is induced, myofibroblasts use both integrin α3β1 and α6β4 as receptors for laminin-332, and become resistant to anoikis via Akt phosphorylation (laminin-332 dependent) and Rac1 activation (laminin-332 independent). Moreover, integrin β4 neoexpression may inhibit myofibroblast aggregation through downregulation of β-catenin.
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