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

Abstract

IntroductionAlthough 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.MethodsThree 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.ResultsMDA-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.ConclusionInvasive 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.

System-Wide Analysis Reveals a Complex Network of Tumor-Fibroblast Interactions Involved in Tumorigenicity

“…Strikingly, the top four pathways identified by gene-set enrichment analysis (GSEA) that are activated by exposure of tumor-promoting human fibroblasts to breast cancer cells are also amongst the top ten pathways activated in cultures of patient-derived breast carcinoma fibroblasts relative to normal fibroblasts (Figure 1). These pathways are ECM-receptor interaction, focal adhesion, integrin signaling and integrin cell-surface interactions; interrelated pathways that have been shown to be involved in the activation of cancer associated fibroblasts [21], [24], [25]. Additionally, most of the other top ten activated pathways in both systems were activated in the other system at lower ranking but still significant levels.…”

Cellular organization and molecular differentiation model of breast cancer-associated fibroblasts

“…Fibroblasts are likely to be more restricted in their differentiation potential than multipotent MSCs, but they may retain some cellular plasticity which appears to be regulated by TGFβ. Very few studies have reported an anoikis-resistant phenotype of fibroblasts, however with a protective role of TGFβ on survival of differentiated fibroblasts [24, 25] which may be a distinct functional feature compared to assessing single-cell survival with subsequent sphere formation. Notably, CAFs appear to be associated with a loss in sphere-forming capacity potentially representing a more differentiated cell type with diminished regenerative ability or cellular plasticity.…”

Laminin-511

“…The predominance of LM-511 in advanced breast tumors however does not preclude a contribution by other LM isoforms during the early stage of tumor progression. For instance, the presence of LM-332-producing myofibroblasts at the tumor-stromal interface has been demonstrated in some invasive breast tumors 49 , 50 . LM-332 is expressed also in TN tumors 39 but it is not clear whether these represent a subset of breast cancer patients distinct from those with LM-511-expressing tumors.…”

“…For example, downregulation of LM-511 and LM-332 occurring in cells undergoing EMT 78 , 79 may be necessary to decrease the strength of adhesion and facilitate the early acquisition of a mesenchymal phenotype. Cells migrating away from the primary tumor, could utilize myofibroblast-derived LM-332 at the tumor-stroma interface 50 . Conversely, re-expression of LM-511 at late stage of metastasis may provide the plasticity required for the reverse process of MET and facilitate the re-establishment of a proliferative epithelial phenotype at metastatic sites.…”