Polarity protein Canoe mediates overproliferation via modulation of JNK, Ras‐MAPK and Hippo signalling

Abstract Objectives Over the past decade an intriguing connection between cell polarity and tumorigenesis has emerged. Multiple core components of the junction complexes that help to form and maintain cell polarity display both pro‐ and anti‐tumorigenic functions in a context‐dependent manner, with the underlying mechanisms poorly understood. Materials and Methods With transgenic fly lines that overexpress or knock down specific signalling components, we perform genetic analysis to investigate the precise role of the polarity protein Canoe (Cno) in tumorigenesis and the downstream pathways. Results We show that overexpression of cno simultaneously activates JNK and Ras‐MEK‐ERK signalling, resulting in mixed phenotypes of both overproliferation and cell death in the Drosophila wing disc. Moderate alleviation of JNK activation eliminates the effect of Cno on cell death, leading to organ overgrowth and cell migration that mimic the formation and invasion of tumours. In addition, we find that the Hippo pathway acts downstream of JNK and Ras signalling to mediate the effect of Cno on cell proliferation. Conclusions Our work reveals an oncogenic role of Cno and creates a new type of Drosophila tumour model for cancer research.


| INTRODUC TI ON
Cell polarity is the morphological and functional asymmetry that helps to form tissue structures such as tubes and alveoli. 1,2 This type of asymmetric distribution of intracellular proteins is established and maintained mainly via cell-cell junctions and adhesions that are formed by a diversity of polarity proteins and adhesion molecules. 3,4 Among these are three groups of core regulators of cell polarity, including the apical Crumbs polarity complex that consists of Crumbs (Crb)/Pals/Patj, the basolateral Par polarity complex that consists of Cdc42/Par3/Par6/aPKC and the Scribble polarity complex that consists of Scribble (Scrib)/Discs large (Dlg)/Lethal (2) giant larvae (Lgl). 5,6 Over the last decade, evidence has been accumulating for the link between tumorigenesis and defects in cell polarity. For instance, in mammary epithelial cells, depleting Crb or Pals caused dephosphorylation and nuclear translocation of YAP, which is a crucial step for the inhibition of the Hippo signalling pathway and results in increased cell proliferation. 7,8 In a DMBA/TPA-induced skin cancer model, the loss of Par3, aPKC or both, strongly reduced tumour size and multiplicity, mainly via impairing the activation of ERK1/2 and Akt by Ras. 9 In mammary epithelia, dysregulation of Scrib prevented Myc-induced apoptosis and promoted epithelial-mesenchymal transition (EMT) and tumorigenesis. 10,11 The effects of cell polarity disruption on tumour growth rely on the association and regulation of downstream signalling pathways. 12,13 Besides, the roles of polarity proteins in tumorigenesis seem complicated, displaying either pro-or anti-tumorigenic functions and largely depending on the context of the cells. 5,13,14 The adhesion molecule Afadin (AF-6), encoded by theMLLT4 gene, is localised at cell-cell adhesion sites in epithelial cells and fibroblasts to help the formation of adherens junctions (AJs) and maintain cell polarity. [15][16][17] Originally identified as a fusion partner of the MLL gene in acute myeloid leukaemia with chromosome translocation, 18   is also associated with initiation and progression of solid tumours.
Low levels of AF-6 expression was reported in 15% of breast cancer patients and linked to adverse prognosis, 19 and the loss of AF-6 was found to promote pancreatic cancer metastasis by inducing Snail expression, 20 suggesting that AF-6 might be a tumour suppressor. In another study, however, elevated AF-6 expression was closely related to adverse outcomes of breast cancer patients. 21 Furthermore, phosphorylation of AF-6 by Akt induced its translocalisation from AJs to the nucleus and increased breast cancer cell migration, 22 revealing the pro-tumorigenic role of AF-6. Therefore, the precise roles of Afadin in tumorigenesis and the underlying mechanisms still remain unclear. As a classic model organism for the research of developmental biology, Drosophila has been recently used in cancer studies and provides great insights into the understanding of tumour initiation and progression. 23,24 Indeed, the first in vivo evidence for the contributions of polarity proteins to tumorigenesis came from the study of Drosophila brains, in which the cells with mutant polarity genes dlg or lgl displayed overgrown and invasive behaviours. 25,26 Subsequent studies demonstrated that loss of scrib, dlg or lgl in the larval eye disc accelerated the growth and metastasis of ras V12 -induced benign tumours, which was largely dependent on the activation of the JNK signalling pathway. 27,28 In addition to this ras V12 -polarity defects model, other Drosophila models for tumorigenesis have also been developed. For example, co-overexpression of EGFR and PI3K in larval glia induced neoplasia in Drosophila and mimicked glioma. 29,30 Besides, elevating the levels of the Src kinase in cells along the anterior-posterior (A/P) compartment boundary of Drosophila wing disc produced a metastatic phenotype, providing a model for genetic screening of genes involved in cancer metastasis. 31,32 Significantly, the core components of the Hippo signalling pathway, which plays vital roles in organ size control and exhibits various mutations in a plethora of tumours, were initially identified using Drosophila genetic models, highlighting the reliability of Drosophila models for cancer research. 8,33 In this study, we show that the overexpression of Cno, the Drosophila homolog of AF-6, induces cell proliferation, cell death and cell migration in the larval wing disc. We find that these mixed effects result from strong activation of JNK signalling and Ras-MAPK signalling. Moderately reducing the activation levels of JNK signalling suppresses the effect of Cno on cell death, thereby inducing massive cell overproliferation and disc overgrowth. In addition, we demonstrate that Hippo signalling acts as a downstream effect or to mediate Cno-induced proliferation, revealing the underlying mechanism for the pro-tumorigenic function of Cno in Drosophila.

| TUNEL staining
Staining was carried out according to the protocol provided by the manufacturer (MK1021, Boster). Briefly, wing discs from third instar larva were dissected and fixed with 4% formaldehyde in 10 m mol L −1 PBS at room temperature for 30 minutes and washed with PBS and distilled water, respectively. Discs were then incubated with labelling buffer in a wet box at 37°C for 2 hours and washed with 0.01 M TBS. After blocking at room temperature for 30 minutes, discs were incubated with anti-Digoxin at 37°C for 30 minutes. Washed with TBS, discs were reacted with diluted SABC at 37°C for 30 minutes.
After washing with TBS, discs were mounted for microscopy.

| Immunostaining and image acquisition
Staining was carried out using standard protocols. Briefly, wing discs from third instar larva were dissected and fixed in PBS-T containing 4% formaldehyde for 20 minutes at room temperature. After washing with PBS-T, they were blocked with 5% BSA in PBS-T for 30 minutes and then incubated with primary antibodies overnight at 4°C. Washed discs were subsequently incubated with secondary antibodies. The following primary antibodies were used: mouse anti-MMP1 (3A6B4, 1:300) and mouse anti-Wg (4D4, 1:500) were purchased from Developmental Studies Hybridoma Bank. Mouse anti-β-galactosidase (sc-65670, 1:500) was from Santa Cruz. Rabbit anti-Caspase3 (#9661, 1:1000) and rabbit anti-p-Histone H3 (#9701, 1:1000) was from Cell Signaling Technology.
Secondary antibodies goat anti-mouse Alexa Fluor594 (A11012, 1:1000) and goat anti-rabbit Alexa Fluor594 (A11005, 1:1000) were from Invitrogen. Fluorescence images were recorded using a Nikon DS-Ri1 fluorescence microscope and a Zeiss LSM 880 confocal microscope. Images of the adult eyes were acquired using a Nikon SMZ-745T trinocular stereo microscope. Images were then analysed using Zeiss Zen, Image J and Adobe Photoshop software.

| Overexpression of cno induces mixed effects in the wing disc
To explore the role of Drosophila AF-6 in cell proliferation, we overexpressed cno at the A/P compartment boundary of Drosophila wing disc by ptc-Gal4. The expression domain of cno, labelled by GFP-positive cells, became broader and irregular than the control, with some cells migrating into the posterior compartment of the disc ( Figure 1A,B, Supporting Information Figure   S1D). We monitored the signal of the mitosis marker, phosphorylated Histone H3 (pH3) and observed a significant increase in the number of pH3-positive cells in the region of cno overexpression in the wing disc (Supporting Information Figure S1 A-C). The activation of JNK signalling has a well-established role in inducing cell migration in Drosophila wing disc. [34][35][36] We therefore examined the expression of JNK target genes in these wing discs. Significantly

| Moderate alleviation of JNK activation augments Cno-mediated proliferation
To investigate the roles of the JNK pathway in Cno-mediated phenotypes in Drosophila wing disc, we examined the effects of differentially reducing the levels of Cno-induced JNK activation. hemipterous signalling, while the knockdown of bsk inhibits it more effectively.
As suggested by the expression domain of GFP, the knockdown of hep or bsk at the A/P compartment boundary had no effect on cell proliferation or migration (Supporting Information Figure S3).
Interestingly, cno overexpression in the presence of hep knockdown at the A/P compartment boundary induced massive cell proliferation and disc overgrowth, with the GFP-positive cells spreading throughout the wing disc (Figure 2A,B, Supporting Information Figure S4).
The combination with bsk knockdown displayed similar but weaker phenotype ( Figure 2C). Similar effects by the knockdown of hep or bsk were also observed when cno was overexpressed in other compartments of the wing disc (Supporting Information Figure S5). The  Figure S6).

| Cno induces proliferation via the Hippo signalling pathway
The Hippo signalling pathway is an evolutionarily conserved signal transduction pathway that plays vital roles in cell proliferation and apoptosis to control cell fate, organ size and tissue homeostasis. 8    To investigate whether the Ras-MAPK pathway is involved in Cnoinduced overproliferation, we examined the effects of ras knockdown in above genetic setups. As controls, the knockdown of ras at the A/P compartment boundary or the dorsal compartment of the wing disc showed no effect on cell proliferation (Supporting Information Figure S7). In contrast, cell overproliferation induced by the overexpression of cno, in the absence or presence of hep knockdown, was strongly inhibited by the knockdown of ras ( Figure 5A-H).

| The Ras-MAPK signalling pathway regulates Cno-induced cell proliferation
Intriguingly, despite the effective inhibition of cell overproliferation, the knockdown of ras seemed unable to block cell migration induced by cno overexpression as was achieved by bsk knockdown ( Figure 2C and Figure 5A-H). These data suggest that Cno-induced phenotypes can be partially separated and are mediated by distinct pathways. Whereas, the Ras-MAPK pathway specifically contributes to the regulation of cell proliferation, the JNK pathway regulates both cell proliferation and migration. It has been reported that the Ras-MAPK signalling influences cell growth through an Yki-dependent mechanism. 46 We thus examined whether the effects of ras  Figure S8). Together, our results suggest that Cno can activate both JNK and Ras-MAPK signalling and regulate cell proliferation via the downstream Hippo signalling.

| D ISCUSS I ON
Although dual regulation of cell proliferation by polarity proteins have been observed and studied for quite a period, controversies about the precise roles of cell polarity in such an important cellular process still exist. 13 For instance, the adhesion molecular Afadin, which contributes to forming and sustaining cellular junctions and cell polarity, has been reported to have pro-or anti-tumorigenic functions. 47 In this work, by expressing Cno, the Drosophila homolog of Afadin, we observed mixed phenotypes of cell death, growth and migration. Since the JNK signalling pathway is well known to participate in all of these cellular processes, 34

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.

AUTH O R CO NTR I B UTI O N S
HS designed the research; ZM, PL and XH performed the experiments; HS and ZM analysed the data and wrote the paper.