Elsevier

Cellular Signalling

Volume 26, Issue 9, September 2014, Pages 1765-1773
Cellular Signalling

Lysyl oxidase-like 2 (LOXL2) controls tumor-associated cell proliferation through the interaction with MARCKSL1

https://doi.org/10.1016/j.cellsig.2014.05.010Get rights and content

Highlights

  • LOXL2 promotes cell metastasis by inhibiting MARCKSL1-stimulated apoptosis.

  • LOXL2 activates cell growth by inhibiting MARCKSL1-induced apoptosis.

  • LOXL2 activates the phosphorylation of FAK/Akt/mTOR signaling cascade components.

  • MARCKSL1 suppresses LOXL2-stimulated breast tumorigenesis.

Abstract

Lysyl oxidase-like 2 (LOXL2) is a member of the lysyl oxidase gene family that contributes to the invasiveness and metastasis in tumor progression. However, the role of LOXL2 in cellular signaling is incompletely understood. In this study, we investigated a possible mechanism of LOXL2 function in tumor metastases in vitro, using a human breast carcinoma cell line. Myristoylated alanine-rich C kinase substrate-like 1 (MARCKSL1), a modulator in the regulation of cellular homeostasis, was identified as a LOXL2 interacting protein. We examined the binding domains that are required for the interaction between LOXL2 and MARCKSL1. The scavenger-receptor domain of LOXL2 was shown to interact with the N-terminal domain of MARCKSL1. Luciferase activity was noticeably reduced by the transfection of MARCKSL1 in a dose-dependent manner. In addition, over-expression of LOXL2 activates cell growth by inhibiting MARCKSL1-induced apoptosis. The effect of LOXL2 on cell cycle and apoptosis-related components was also confirmed through the silencing of LOXL2 expression. LOXL2 activates the FAK/Akt/mTOR signaling pathways, and MARCKSL1 suppresses LOXL2-induced oncogenesis. These insights supply evidence that LOXL2 promotes cell proliferation and inhibits apoptotic cell death. Taken together, our results indicate an underlying mechanism for an increase of LOXL2-related activity in breast tumor cells.

Introduction

Lysyl oxidase-like 2 (LOXL2) is a member of the lysyl oxidase (LOX) family, which has a highly conserved carboxyl C-terminal domain that contains a copper-binding site, and a lysyl/tyrosyl quinine (LTQ) group, that are both essential for catalytic activity [1]. In contrast with the other members of this family, LOXL2 contains four scavenger receptor cysteine-rich (SRCR) domains thought to be involved in protein–protein interactions at the N-terminal region [2] and in ligand binding of both soluble proteins and membrane-bound protein receptors [3], [4]. In addition, LOXL proteins are currently thought to be involved in developmental regulation, cell adhesion, senescence, cell migration and invasion, metastases, epithelial–mesenchymal transition (EMT) and malignant transformation [2], [5], [6], [7]. Mechanistically, the role of LOXL2 in the metastasis of gastric tumors involves the activation of the Snail1/E-cadherin and FAK/Src signaling pathways, although activation of the FAK/Src signaling cascade by secreted LOXL2 seems to be responsible for the stimulation of gastric cell invasion and metastasis [8]. Generally, metastatic cancers are found at the same time as the primary malignancy. The metastatic process comprises a complex series of steps, which are required for the malignant cells to leave the original cancer site. These steps include a loss of cell–cell adhesion, increased cell motility, migration to other locales of the body via the bloodstream, and invasion into the surrounding tissues. This is associated with processes involved in the epithelial to mesenchymal transition, which contributes to the widespread metastasis of epithelial cancer cells [9], [10], [11].

Integrins are transmembrane adhesion receptors that regulate cell-to-cell contact, as well as serve as factors in adhesion of the extracellular matrix, while also playing critical roles in cell–cell adhesion, metastasis, proliferation, migration, tumor progression, and programmed cell death as a part of cellular biological homeostasis [12]. The extracellular matrix plays vital roles in several biological processes, such as migration, protein expression, morphology, proliferation, and differentiation [13]. Specifically, αV integrins can induce activation of focal adhesion kinase (FAK) leading to the activation of Src kinase, and thereby affecting angiogenesis [14]. FAK is recruited at an early stage to focal adhesions, where it acts as a tyrosine kinase to initiate downstream signaling and has recently been shown to modulate tumor progression. It is induced by focal adhesion complex-related growth factors, and is constitutively associated with β-integrin subunits of the receptors. FAK is a pivotal protein tyrosine kinase, which controls various aspects of integrin signaling through phosphorylation. FAK is associated with endothelial cell motility, survival, cell cycle progression and migration, is increased in many tumor types, and is highly conserved [15]. The over-expression of LOXL2 has been detected in various human malignancies, including breast cancers [16], [17], [18], colorectal adenocarcinomas [18], [19], gastric cancers [8], [20], hepatocellular carcinomas [18], [20], head and neck squamous cell carcinomas [21], lung squamous cell carcinoma [18], [22], and pancreatic carcinomas [18], [20], [23].

Members of the myristoylated alanine-rich C kinase substrate (MARCKS) family play an important role in actin/cytoskeletal regulation, protein kinase C (PKC) signaling and Ca2 +/calmodulin signaling pathways in many mammalian tissues [24], [25]. Myristoylated alanine-rich C kinase substrate-like 1 (MARCKSL1) is a member of the MARCKS family and is a major cellular substrate for PKC as a membrane-bound protein. MARCKSL1 (also known as MacMARCKS, MLP, MRP, or F52) has been implicated in the regulation of integrin activation, brain development, cell migration, and cell adhesion, as well as phagocytosis, membrane traffic, mitogenesis, and endocytosis [26], [27], [28], [29], [30], [31]. Li et al., [32] have reported that MARCKS is a target of miR-21 a micro-RNA that enhances invasion and metastasis in a number of human tumors indicating it is an important tumor suppressor. Recent studies reported that JNK phosphorylation of MARCKSL1 controls actin homeostasis and migration under physiological conditions in neurons and when ectopically expressed in prostate cancer cells [31].

In this study, we begin the characterization of LOXL2-dependent signaling pathways, using a yeast two-hybrid system as a means to screen a human cDNA library for novel LOXL2 binding proteins. We show that the binding of LOXL2 and MARCKSL1 modulates cellular biological functions. We also validated MARCKSL1-induced apoptotic features found in breast tumor cells are affected by LOXL2 signaling pathways by showing that the pattern of expression for the apoptosis-associated proteins are regulated by siRNA inhibition of LOXL2 expression. Based on these observations, we suggest that one possible cellular molecular mechanism of LOXL2 function is preventing apoptosis induced by the tumor suppressor gene MARCKSL1.

Section snippets

Cell lines, cell culture, and antibodies

Human breast MDA-MB-231 cells and HEK 293T cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA). Cells were maintained in accordance with ATCC recommendations. Wortmannin and LY294002 chemicals were obtained from Sigma (St. Louis, MO). The primary antibodies used in this study were anti-LOXL2, anti-MARCKSL1, anti-pro-caspase-3, anti-pro-PARP, anti-cyclin D1, anti-CDK4, anti-p21, anti-p27, anti-Bcl-2, anti-p53, anti-phospho-Src(Y416), anti-Src,

Physical interaction of LOXL2 with MARCKSL1

To address the physiological relevance of LOXL2 in tumor metastasis, we carried out a yeast two-hybrid and co-immunoprecipitation assay to identify potential LOXL2-interacting proteins. The human cDNA library was fused to the transcription activator, pJG4-5 vector and was transformed into yeast cells containing pGilda-LOXL2 as bait. Approximately 5.5 × 106 independent transformants were pooled. After re-spreading on selection media (Ura-, His-, Trp-, Leu-), we obtained six positive colonies. All

Discussion

Previous studies have shown that higher LOXL2 expression is associated with invasiveness of breast carcinoma cells and lower survival of breast cancer patients [37]. However, it is unknown whether LOXL2 directly affects the metastasis and progression of cancer in breast tumorigenesis. The present study, provides a new evidence that LOXL2 promotes metastasis by enhancing LOXL2-dependent FAK/Akt/mTOR phosphorylation. First, higher LOXL2 expression activated cellular metastasis by inhibiting

Acknowledgments

We thank Dr. S. A. Martinis (Department of Biochemistry, University of Illinois at Urbana-Champaign, IL, USA), and Richard Yoo (University of Washington, Seattle, WA, USA) for critical reading of the manuscript. This work was supported by grants from the National Cancer Center, Korea (NCC-1410312-1 and NCC-1410311-1).

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    These authors contributed equally to this work.

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