Lysyl oxidase-like 4 exerts an atypical role in breast cancer progression that is dependent on the enzymatic activity that targets the cell-surface annexin A2

Abstract

BackgroundLOX family members are reported to play pivotal roles in cancer. Unlike their enzymatic activities in collagen cross-linking, their precise cancer functions are unclear. We revealed that LOXL4 is highly upregulated in breast cancer cells, and we thus sought to define an unidentified role of LOXL4 in breast cancer.MethodsWe established the MDA-MB-231 sublines MDA-MB-231-LOXL4 mutCA and -LOXL4 KO, which stably overexpress mutant LOXL4 that loses its catalytic activity and genetically ablates the intrinsic LOXL4 gene, respectively. In vitro and in vivo evaluations of these cells’ activities of cancer outgrowth were conducted by cell-based assays in cultures and an orthotopic xenograft model, respectively. The new target (s) of LOXL4 were explored by the MS/MS analytic approach.ResultsOur in vitro results revealed that both the overexpression of mutCA and the KO of LOXL4 in cells resulted in a marked reduction of cell growth and invasion. Interestingly, the lowered cellular activities observed in the engineered cells were also reflected in the mouse model. We identified a novel binding partner of LOXL4, i.e., annexin A2. LOXL4 catalyzes cell surface annexin A2 to achieve a cross-linked multimerization of annexin A2, which in turn prevents the internalization of integrin β-1, resulting in the locking of integrin β-1 on the cell surface. These events enhance the promotion of cancer cell outgrowth.ConclusionsLOXL4 has a new role in breast cancer progression that occurs via an interaction with annexin A2 and integrin β-1 on the cell surface.

Lysyl oxidase-like 4 promotes the invasiveness of triple-negative breast cancer cells by orchestrating the invasive machinery formed by annexin A2 and S100A11 on the cell surface

“…Bio-PLG added to the MDA-MB-231 cell culture was co-localized with S100A11 in most parts of the cell membrane before fixation treatment of the cells, which was also confirmed ( Figure 2B ). It was confirmed that the PLG attached to the plasma membrane surface leads to plasmin activity, which is much higher in parental cells than in the LOXL4 KO cells ( Figure 2C ) ( 7 ). In addition, the manifestation of enzymatic active-form plasmin from the PLG in the membrane fraction required S100A11 since siRNA-mediated lowered expression of S100A11 markedly obstructed the plasmin activity ( Figure 2D ).…”

“…The multimerized annexin A2 also coordinates with abundant integrin-b1 on the cell surface, allowing the growth, migration, and invasion of TNBC cells. The newly identified machinery of plasmin activation works cooperatively with the integrin-b1-mediated pathway we formerly revealed (7), particularly for the invasion event.…”

“…The stable transformant overexpressing the LOXL4 wild type originating from MDA-MB-231 cells (named MDA-MB-231 LOXL4 wt) has been reported previously (6). The MDA-MB-231 LOXL4 knock-out (KO) subline was also the same as that previously reported (7). All cell lines were cultivated in DMEM/F12 medium (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS).…”

“…Our recent efforts have shown that lysyl oxidase-like 4 (LOXL4) may be a potent new target for TNBC treatment ( 6 , 7 ). LOXL4 is a secretory lysyl oxidase enzyme and one of the five members of the LOX family of proteins (LOX; LOXL1–4); these proteins enable cross-bridging among extracellular matrices such as collagen and are closely involved in the formation of cancer-growing stroma ( 8 – 10 ).…”

“…In our study, we identified a new role of the LOXL4 secreted in TNBC progression: in addition to its traditional collagen cross-linking function, it also targets cancer cell surface annexin A2 as a substrate for enzymatic cross-linking modification. The LOXL4-mediated cross-bridging involves annexin A2 polymerization on the membrane, with the polymerized protein in turn binding to integrin-β1, eventually leading to cell surface accumulation of integrin-β1 at a significant level ( 7 ). The process plays a critical role in cancer cell adhesion to tissues, enabling accelerated proliferation and invasion.…”