Secreted AGR2 promotes invasion of colorectal cancer cells via Wnt11-mediated non-canonical Wnt signaling

Highlights

• Secreted AGR2 does significantly promote CRC cell migration and metastasis.

• Secreted AGR2 elevates the expression of Wnt11 in CRC.

• Secreted AGR2 promotes CRC migration and metastasis through Wnt11-meadied non-canonical Wnt signaling.

• Secreted AGR2 negatively regulates the activity of the canonical β-catenin pathway via activating CaMKII-dependent non-canonical Wnt signaling.

Abstract

Human anterior gradient-2 (AGR2), a member of protein disulfide isomerase (PDI) family, is present in both intracellular and extracellular compartments. Although AGR2 is overexpressed in various human cancers and reported to promote aggressive tumor features, little is known regarding AGR2′s extracellular functions during tumorigenesis. Here, we demonstrate that secreted AGR2 promotes cell migration and metastasis of colorectal cancer (CRC) in vitro and in vivo. Mechanistically, secreted AGR2 elevated Wnt11 expression, triggering non-canonical Wnt signaling: the Ca²⁺/Calmodulin-dependent protein kinase II (CaMKII) and c-jun amino-terminal kinase (JNK) pathways. Knockdown of Wnt11 or pretreatment with CaMKII and JNK inhibitors reversed the secreted AGR2′s migration-promoting effect. Further studies revealed that AGR2 antagonized canonical Wnt/β-catenin signaling via activating CaMKII. Collectively, our study uncovers a critical role of Wnt11-mediated non-canonical Wnt signaling (CaMKII and JNK pathways) in secreted AGR2′s promoted migration of CRC cells. These results raise the possibility that secreted AGR2 may be a potential therapeutic target towards inhibiting CRC metastasis.

Introduction

The human anterior gradient-2 (AGR2) is the orthologue of the Xenopus Anterior Gradient-2 (XAG-2) protein, which is responsible for patterning embryos and regulating the differentiation of cement glands and the expression of neural marker genes [1]. Human AGR2 is a mucin-like molecule predominately found in tissues with mucus-secreting or endocrine cells, such as colon, rectum, stomach, breast, and prostate [2], [3]. AGR2 was originally described in human breast cancer [4]. Since then, it has been investigated in various human cancer types and AGR2′s aberrant expression was reportedly associated with poor clinical outcomes [3]. In normal intestine, AGR2 as a critical modulator of intestinal homeostasis presents within the ER of intestinal secretory epithelial cells and regulates production of intestinal mucins via formation of mixed disulfide bonds [5]. Mice lacking AGR2 expression are unable to produce intestinal mucins and highly susceptible to colitis [6]. Further, AGR2 expression is related to inflammatory bowel disease. The mutations in murine AGR2 gene that decrease AGR2 mRNA level are associated with Crohn’s disease as well as ulcerative colitis [7]. However, the clinical significance and potential effects of AGR2 on colorectal cancer (CRC) remain obscure.

As a member of the protein disulfide isomerase (PDI) family, AGR2 intracellularly localizes to endoplasmic reticulum (ER) and involves in protein maturation [8], [9]. Of note, AGR2 can escape ER retrieval machinery and be secreted to extracellular matrix as well as serum and urine, etc [4]. Although AGR2 overall overexpression enhances cell migration and metastasis in several cancers [3], the underlying mechanisms remain elusive partly because most of these studies did not individually investigate the roles of intracellular AGR2 and secreted AGR2 in metastasis. Recently, tumor-secreted AGR2 was reported to promote migration of vascular endothelial cells and fibroblasts. However, whether secreted AGR2 promotes CRC cell migration as well as the molecular mechanisms is not clear [10].

Deregulation of Wnt signaling is a critical molecular event during CRC tumorigenesis [11]. Wnt signaling is activated by multiple Wnt ligands and signals through either the canonical pathway or the non-canonical pathways [12]. The prominent feature of the “canonical” pathway is to employ β-catenin as a transcriptional coactivator [13]. Deregulation of this pathway occurs in the vast majority (80–90%) of CRC [14]. Without the presence of Wnt ligands, cytosolic β-catenin is phosphorylated for degradation by a destruction complex containing adenomatous polyposis coli (APC), Axin, glycogen synthase kinase 3β (GSK3β) and other proteins. Once bound by Wnt ligands (such as Wnt1 and Wnt3a), the Fz/LRP coreceptor complex disrupts the aforementioned destruction complex, allowing β-catenin accumulation and nuclear translocation. In nuclei, β-catenin works together with the T-cell factor/lymphoid enhancer factor (TCF/LEF) family, regulating the expression of downstream genes [15], [16]. In contrast, other Wnt ligands, such as Wnt5a and Wnt11, preferentially trigger non-canonical pathways that function in β-catenin-independent manners [17]. Depending on the downstream molecules, the non-canonical pathways mainly refer to these two: the Wnt/Ca²⁺ pathway, and the planar cell polarity (PCP) pathway. In the Wnt/Ca²⁺ pathway, calmodulin-dependent protein kinase II (CaMKII) along with Protein kinase C (PKC) stimulates target gene expression through the nuclear factor of activated T cell (NFAT). In the PCP pathway, c-Jun N-terminal Kinase (JNK) acts as the downstream effector regulating target gene expression via interacting with activator protein 1 (AP-1) [13], [18]. Wnt signals are context-dependently transduced to canonical or non-canonical pathways. Intriguingly, previous studies revealed an important finding that non-canonical Wnt signaling can function to negatively modulate canonical Wnt/β-catenin signaling [19].

Here, we report that secreted AGR2 significantly promotes the migration of CRC cells in vivo and in vitro. This is mechanistically because secreted AGR2′s upregulates Wnt11 that activates JNK and CaMKII pathways, which in turn promotes CRC cell migration/ invasion and β-catenin degradation, suppressing canonical Wnt signaling.