Lysyl oxidase-like 2 (LOXL2) controls tumor-associated cell proliferation through the interaction with MARCKSL1
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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LOXL2 in cancer: regulation, downstream effectors and novel roles
2020, Biochimica et Biophysica Acta - Reviews on CancerCitation Excerpt :In human breast carcinoma cell lines, LOXL2 interacts with myristoylated alanine-rich C-kinase substrate (MARCKS)-like protein 1 (MARCKSL1) through its N-terminal SRCR domain. LOXL2 promotes cancer cell growth by inhibiting apoptosis induced by MARCKSL1, which in turn, inhibits LOXL2-mediated FAK/AKT/mTOR signaling cascades [22]. LOXL2 decreases protease and proteasome-mediated protein degradation of integrin α5 (ITGA5) and integrin β1 (ITGB1) in ccRCC [28].
Evolving roles of lysyl oxidase family in tumorigenesis and cancer therapy
2020, Pharmacology and TherapeuticsCitation Excerpt :Elevated expression of LOXL2 promoted the tumor fibrosis and facilitated tumor cells to invade blood vessels, nerves, and muscles adjacent to the breast cancer (Akiri et al., 2003). LOXL2 facilitated the proliferative ability and suppressed the apoptotic ability of the breast cancer cells, and the LOXL2-mediated tumorigenesis can be inhibited by the myristoylated alanine-rich C kinase substrate-like 1 (MARCKSL1) (Kim et al., 2014). Dormant tumor cells (DTC) acquired cancer stem cell (CSC)-like phenotype under the expression of LOXL2, which promoted their conversion to proliferative outgrowth in recurrent breast carcinomas (Weidenfeld et al., 2016).
Emerging Roles of Extracellular Hsp90 in Cancer
2016, Advances in Cancer ResearchCitation Excerpt :LOXL2 is a member of the lysyl oxidase family of proteins, which modify the ECM by cross-linking collagen. Cross-linking alters the stiffness of the matrix, resulting in phenotypic changes such as increased cell motility and proliferation (Barker, Bird, Lang, & Erler, 2013; Kim et al., 2014). Increased expression of LOXL2 by cancer cells has been associated with increased invasion (Moon et al., 2013) and poor clinical prognosis (Ahn et al., 2013; Barker et al., 2011).
LOXL2 in Cancer: A Two-Decade Perspective
2023, International Journal of Molecular Sciences
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These authors contributed equally to this work.