APLP2 Modulates JNK-Dependent Cell Migration in Drosophila

Amyloid precursor-like protein 2 (APLP2) belongs to the APP family and is widely expressed in human cells. Though previous studies have suggested a role of APLP2 in cancer progression, the exact role of APLP2 in cell migration remains elusive. Here in this report, we show that ectopic expression of APLP2 in Drosophila induces cell migration which is mediated by JNK signaling, as loss of JNK suppresses while gain of JNK enhances such phenotype. APLP2 is able to activate JNK signaling by phosphorylation of JNK, which triggers the expression of matrix metalloproteinase MMP1 required for basement membranes degradation to promote cell migration. The data presented here unraveled an in vivo role of APLP2 in JNK-mediated cell migration.


Introduction
Amyloid precursor-like protein-2 (APLP2) belongs to the protein family that includes amyloid precursor protein (APP) and amyloid precursor-like protein-1 (APLP1) in mammals [1,2]. The three proteins show sequence similarity in the extracellular E1, E2 and the intracellular domains, while only APP and APLP2 share a special Kunitz protease inhibitor (KPI) domain and an Asp-Glu-rich domain, suggesting a role that is likely specific for the two proteins [3,4]. Consistent with this notion, APP and APLP2 are widely expressed in many tissues, whereas APLP1 is predominantly restricted to the neural cells [5][6][7]. Studies in knockout mice have unraveled that all the single knockout and the APP/APLP1 double knockout mice are viable and fertile displaying no evident phenotype, while the double knockout mice of APP/APLP2 or APLP1/APLP2 are prenatally lethal, implying a specific role of APLP2 in animal development [8][9][10], which is consistent with their divergent protein interaction networks observed in an in vivo brain study [11]. Furthermore, the phylogenetic tree of APP protein family indicates that APLP2 is more distant from an inferred ancestral gene than APP and APLP1 [12], suggesting APLP2 may perform distinct in vivo functions.
APLP2 has been shown to regulate multiple cellular functions such as neurite outgrowth, axogenesis, corneal epithelial wound healing, cell adhesion, migration [13], and mitosis [14][15][16][17]. The expression level of APLP2 is upregulated in the pancreatic tumor cell lines S2-013, the prostate cancer cell line DU145, and certain human cancers such as breast cancer [18][19][20] but is downregulated in the lymphoma cell lines [21] and in the lung neuroendocrine tumors [22]. Thus, the exact role of APLP2 in tumorigenesis remains elusive.
The c-Jun N-terminal Kinase (JNK) pathway is a highly conserved signaling from Drosophila to human that governs diverse cellular functions including cell proliferation, differentiation, death, and migration and regulates physiological processes such as stress response and lifespan [23][24][25][26][27][28]. However, an in vivo role of APLP2 in modulating JNK signaling has not been characterized.
In this work, we studied the in vivo function of human APLP2 in Drosophila. We found that ectopic expression of APLP2 in the wing disc of 3 rd instar larvae promotes cell migration, which is suppressed by loss of JNK signaling while exacerbated by gain of JNK signaling. Consistently, APLP2 activates JNK signaling by the phosphorylation of JNK and thus elevates JNK target gene MMP1 expression to initiate 2 BioMed Research International cell migration. This work, therefore, provides the first in vivo function of APLP2 in JNK-mediated cell migration.

Statistical Analysis for Cell Migration.
More than 20 wing discs were dissected for each genotype. The number of migrating cells in the posterior compartment of the wing discs was counted. Unpaired t test by GraphPad Prism 5 was used to analyze the statistical significance. Error bars mean ± SEM, * * * : p<0.001, * * : p<0.01, and n.s.: no significant difference.

APLP2 Promotes Cell Migration in Drosophila.
The expression level of APLP2 is increased in many tumor cells suggesting that APLP2 may play a vital role in tumor formation and metastasis [13,33,34]. To examine the exact function of APLP2 in cell migration in vivo, we ectopically expressed APLP2 along the anterior/posterior (A/P) compartment boundary in 3 rd instar larval wing discs, which has been commonly used to investigate the migrating phenotype in vivo [35]. We noticed that patched-Gal4 (Figures (A) [32,[36][37][38][39][40]. Consistently, RNAi-mediated depletion of puckered (puc), a negative regulator of JNK signaling [41,42], promoted a cell migrating phenotype (Figures (C)-(C"), and (E)). These data indicate that APLP2 is able to trigger cell migration in vivo, which phenocopies that of JNK activation.

JNK is Required for APLP2-Induced Cell Migration.
Since expression of APLP2 induced a migrating phenotype mimicking JNK activation in the wing disc, we hypothesized that JNK signaling pathway might be required for APLP2triggered cell migration. To test this, we first elevated JNK signaling by deleting one copy of the endogenous puc gene encoding a JNK phosphatase that negatively regulates JNK activity [41,42]. Compared with the ptc-Gal4 control (Figures (A) and (I)), we observed that APLP2-induced cell migration phenotype (Figures (B) and (I)) was dramatically enhanced in heterozygous puc E69 (Figures (C) and (I)) or puc H246 (Figure S ) mutants, while neither mutant alone could produce any migration phenotype [43], suggesting a genetic interaction between APLP2 and the JNK signaling in promoting cell migration. To further probe the role of JNK signaling in APLP2-induced cell migration, we blocked JNK pathway by expressing a dominant negative form of Drosophila JNK, Bsk [44], or the JNK phosphatase puc. We found that APLP2-induced cell migration was significantly suppressed by the expression of Bsk DN or puc but remained unaffected by the expression of LacZ (Figures (D)-(F), and (I)). Furthermore, blocking JNK signaling also inhibited APLP2-induced, puc E69 -enhanced cell migration phenotype (Figures (G), (H), and (I)). Thus, we conclude that APLP2 induces JNK-dependent cell migration in vivo.

APLP2 Triggers JNK Activation In Vivo.
The above data suggest that APLP2 promotes JNK-mediated cell migration in vivo. To investigate whether APLP2 is able to activate JNK signaling, we checked the expression of a puc-LacZ reporter, a commonly used readout of JNK signaling [27], and JNK phosphorylation in the wing disc. We found that APLP2 was sufficient to induce puc-LacZ expression (Figures  (H")). Collectively, the data suggest that APLP2 expression is sufficient to trigger JNK activation in the wing disc.
To investigate whether APLP2 could induce JNK activation in other tissues, we checked the salivary glands where ptc-Gal4 is also expressed. Compared to the control  ( Figure S A), expression of APLP2 induced JNK signaling activation, as revealed by the puc-LacZ expression in the gland (Figures S B). Expression of Bsk DN fully suppressed both the endogenous and the ectopically activated expression of puc-LacZ (Figure S C). Together, the data demonstrate that APLP2 is able to activate JNK signaling in a nontissue specific manner.

APLP2 Induces JNK-Mediated MMP1
Expression. JNKdependent cell migration is mediated by transcriptional upregulation of the matrix metalloproteinase MMP1 [45,46], which is required for the degradation of basement membrane and serves as a hall marker for cell migration behaviors in Drosophila [47][48][49][50]. Consistently, expression of APLP2 driven by ptc-Gal4 induced MMP1 expression in the wing disc (Figures (B)-(B")), which was dramatically suppressed by the expression of Bsk DN (Figures (C)-(C")) or puc (Figures (D)-(D")). Intriguingly, APLP2 induced both autonomous and nonautonomous JNK phosphorylation (Figures (F")) and MMP1 expression (Figures (B")), which have been previously reported for other migrationpromoting genes [37,38]. Hence, APLP2 is able to induce JNK-mediated MMP1 activation, which is necessary for basement membrane degradation and cell migration. Similar results were observed in the P-compartment of wing discs when APLP2 expression was initiated by engrailed-Gal4 (en-Gal4) (Figure S ). Finally, we examined the role of MMP1 in APLP2-induced cell migration. We found that RNAimediated MMP1 depletion impeded APLP2-induced cell migration ( Figure S ). Thus, ectopic expression of APLP2 is able to induce JNK-mediated MMP1 upregulation, which is crucial for basement membrane degradation and cell migration. Actin accumulation is a key hint for the cell migration phenotype [45,46], and APLP2 was shown to modulate actin cytoskeleton in pancreatic cancer cells [13,33,34]. Consistently, we found that APLP2 expression could induce actin polymerization in the wing disc ( Figure S ). APLP2 expression is elevated in certain pancreatic and prostate cancer cells as well as in breast cancer samples, while downregulated in lymphoma cells and lung neuroendocrine tumors, implying a controversial role in tumor progression [13,33,34]. In this study, we investigated the in vivo function of APLP2 in Drosophila wing disc epithelia. Our data indicate that APLP2 is able to promote JNK-dependent cell migration in vivo. Mechanistically, APLP2 activates JNK signaling through the phosphorylation of JNK, which upregulates the expression of MMP1 that is essential for basement membranes degradation and cell migration. Our previous work showed that expression of APLP2 could induce the expression of apoptotic gene hid and apoptosis [51], yet APLP2induced cell migration was not blocked by the expression of baculovirus p35 (Figure S ), indicating APLP2-induced cell migration is independent of apoptosis. Consistent with our in vivo results, Chinese hamster ovary (CHO) cells overexpressing APLP2 exhibit increased chemotaxis toward type IV collagen and fibronectin [16], whereas depletion of APLP2 in pancreatic cancer cells resulted in reduced migration and invasion ability [13,33,34]. Intriguingly, comparable expression of APLP1 [52] triggers stronger cell migration than APLP2 in Drosophila [43], suggesting both amyloid precursor-like proteins can promote cell migration in vivo, albeit at different efficiencies. Consistent with our finding, APLP1 and APLP2 are found to be increased in cancers [13,33,34] and knock-down of APLP2 in pancreatic cancer cells reduced the ability of cell migration [13].Yet it remains to be elucidated whether JNK signaling plays a crucial role in APLP2-induced cell migration and tumor invasion in mammals.

Data Availability
No data were used to support this study.

Conflicts of Interest
The authors declare that they have no conflicts of interest.