Loss of DLG5 promotes breast cancer malignancy by inhibiting the Hippo signaling pathway

Discs Large Homolog 5 (DLG5) plays an important role in the maintenance of epithelial cell polarity. Recent research showed that DLG5 is decreased in Yes-associated protein (YAP)-overexpressing cells. However, the exact relationship between DLG5 and YAP is not clear. In this study, we showed that loss of DLG5 promoted breast cancer cell proliferation by inhibiting the Hippo signaling pathway and increasing nuclear YAP expression. Furthermore, depletion of DLG5 induced epithelial-mesenchymal transition (EMT) and disrupted epithelial cell polarity, which was associated with altered expression of Scribble, ZO1, E-cadherin and N-cadherin and their mislocalization. Interestingly, we first reported that loss of DLG5 inhibited the interaction of Mst1 and Lats1 with Scribble, which was crucial for YAP activation and the transcription of TEA domain (TEAD) family members. In summary, loss of DLG5 expression promoted breast cancer malignancy by inactivating the Hippo signaling pathway and increasing nuclear YAP.


Distribution of DLG5 expression in breast epithelial cells, breast cancer cells, breast cancer tissues and para-cancerous tissues.
To identify the relationship between DLG5 and breast cancer, we examined the expression of DLG5 in breast cancer tissues. DLG5 showed stronger staining in normal mammary tissues than in breast cancer tissues. Low-grade cancer tissues had higher expression of DLG5 than high-grade cancer tissues (Fig. 1A,F). Nearly 76% of stage I breast cancer, 55% of stage II breast cancer, and 23% of stage III breast cancer samples showed moderate or strong DLG5 staining. Of the stage III breast cancer tissues, 50% were weakly positive, and 27% were negative (sTable 2). To summarize, stage I and stage II breast cancer tissues demonstrated reduced DLG5 staining, while stage III tissues showed loss of or reduced DLG5 staining.
Estrogen receptor (ER) and progesterone receptor (PR) status is a biomarker for breast cancer therapy and prognosis 16,17 . DLG5 expression showed a significant positive correlation with ER and PR status in breast cancer.
p53 is a tumor suppressor that is important in cell cycle arrest 18,19 . Ki67 is a well-known diagnostic marker for various cancers; its expression is positively associated with cell growth and reflects cell proliferation 20,21 . We observed stronger DLG5 staining in p53+ tissues than in p53− /+ tissues (Fig. 1D,I; sTable 2). Meanwhile, DLG5 staining was weak or negative in Ki67+ tissues but moderate or strong in Ki67− /+ tissues (Fig. 1E,J; sTable 2). These results suggest that loss of DLG5 expression might be associated with breast cancer cell proliferation. Breast cancer cell lines showed similar results. A non-transformed breast epithelial cell line (MCF10A) and low-grade breast cancer cell lines (MCF7, T47D and BT474) showed higher DLG5 protein and mRNA expression levels than high-grade breast cancer cell lines (MDA-MB-468, MDA-MB-231 and MDA-MB-453). DLG5 expression was higher in breast cancer cell lines of the luminal subtype than in those of the basal-like subtype (Fig. 1K,L,M). Additionally, DLG5 and E-cadherin co-localized at MCF10A cell-cell junctions (Fig. 1N), which further verified the important role of DLG5 in the maintenance of epithelial cell polarity. These results suggest that DLG5 might participate in breast cancer development.
Loss of DLG5 promotes cell proliferation. Classical DLG proteins and ZO subfamily members such as DLG1 22 , DLG3 23 , ZO1, ZO2 and ZO3 24,25 have been reported to be associated with cell proliferation. DLG5 possesses a structure similar to classical DLG proteins and ZO subfamily members 26,27 , suggesting that DLG5 may also play a role in cell proliferation. To prove our hypothesis, we constructed DLG5-knockdown MCF10A and MCF7 cell lines (MCF10A-shDLG5 and MCF7-shDLG5), a DLG5-overexpressing MDA-MB-231 cell line (MDA-MB-231-oxDLG5) and respective negative control (NC) cell lines by lentivirus-mediated shRNA interference ( Fig. 2A). Analyses using a Live Cell Imaging System and by cell counting showed that DLG5-knockdown cells had a higher growth rate than NC cells. After 24 hrs, there were nearly twice as many MCF7-shDLG5 cells as NC cells. After 48 hrs, MCF7-shDLG5 cells were at almost 100% confluence, but NC cells were at approximately 50% confluence. The opposite results were observed in MDA-MB-231-oxDLG5 cells (Fig. 2B,C; supplementary movies 1, 2).
A subsequent study in MCF10A-shDLG5 and MCF7-shDLG5 cells revealed an increased percentage of cells in S phase and G2/M phase and a decreased percentage in G1 phase; conversely, DLG5 overexpression in MDA-MB-231 cells inhibited cell cycle progression (Fig. 2D,E). MCF10A-shDLG5 and MCF7-shDLG5 cells showed a higher rate of BrdU incorporation than NC cells. In contrast, percentage of BrdU positive cells in MDA-MB-231-oxDLG5 cells was lower than that of NC cells (Fig. 2F,G). The results indicate that loss of DLG5 may promote cell proliferation by increasing DNA synthesis during S phase.
We also examined the expression of cell proliferation-associated proteins following a change in DLG5 expression. Loss of DLG5 expression induced the up-regulation of cyclin D1 expression and the significant down-regulation of p21, p27 and p53 expression in MCF10A-shDLG5 and MCF7-shDLG5 cells, while overexpressing DLG5 resulted in reduced cyclin D1 expression and increased p21, p27 and p53 expression in MDA-MB-231-oxDLG5 cells (Fig. 2H).
DLG5 knockdown disrupts breast cell acinar polarity. The morphogenesis of NC and MCF10A-DLG5 cells in 3D culture was studied to determine the exact role of DLG5 in the maintenance of cell polarity. Under 3D culture conditions, MCF10A cells proliferate and organize into spheroids, commonly called "acini" 12 . These acini have a central lumen and are lined by layers of polarized epithelial cells. We cultured MCF10A-shDLG5 cells and NC cells in 3D culture for 16 days. DLG5-knockdown cells failed to form acini or formed acini with disordered morphogenesis compared with NC cells (Fig. 3A,B). E-cadherin exhibited a continuous ring-like distribution on the outer layer of the acini formed by MCF10A-NC cells. DLG5 knockdown induced a partial loss of E-cadherin expression and discontinuous E-cadherin distribution on the outer layer of the acini (Fig. 3C). GM130 is regarded as a biomarker of the orientation of the Golgi apparatus toward the lumen in 3D morphogenesis studies; therefore, GM130 mislocalization represents a change in cell polarity 29 . In response to loss of DLG5 expression, GM130 was translocated to the outside surface of the acini (Fig. 3D). These results indicate that DLG5 plays an important role in the maintenance of cell polarity of breast acini.

Loss of DLG5 induces EMT and promotes cell migration and invasion.
Failure to maintain cell polarity is always accompanied by EMT 30,31 . To investigate the role of DLG5 in EMT in breast cancer cells, we compared the expression of EMT makers in MCF10A-shDLG5, MCF7-shDLG5 and NC cells. We found that DLG5 knockdown altered the expression of certain EMT markers; the expression of the mesenchymal markers N-cadherin and vimentin was increased, that of the epithelial marker ZO1 was decreased, and that of the epithelial marker E-cadherin did not change. In MDA-MB-231-oxDLG5 cells, there was a notable increase in ZO1 and E-cadherin expression and a decrease in N-cadherin and vimentin expression (Fig. 4A). Additionally, ZO1, E-cadherin and N-cadherin localized at both cell-cell junctions and in the cytoplasm after DLG5 knockdown (Fig. 4B). The results indicate that loss of DLG5 might partially induce EMT.
In our experiments, we noticed that MCF7-shDLG5 cells exhibited greater mobility than NC cells (supplementary movies). To identify the role of DLG5 in cell mobility, we performed monolayer wound healing and transwell assays with MCF10A-shDLG5 and MCF7-shDLG5 cells. DLG5 knockdown led to increased cell migration without an effect on cell proliferation (Fig. 4C,D). Loss of DLG5 expression also increased the invasive ability of MCF10A and MCF7 cells compared with NC cells (Fig. 4E,F Loss of DLG5 inactivates the Hippo signaling pathway. Scribble is required for Hippo signaling pathway activity in both Drosophila and mammalian cells 32 . Knockdown of Scribble leads to increases in YAP nuclear localization and TEAD transcription owing to the absence of the interactions of Mst1/2 and Lats1/2 with Scribble, which ultimately inactivates the Hippo signaling pathway 33,34 . ZO2 is also a member of the MAGUK family and is crucial in cell-cell interactions; activation of the PDZ domain in ZO2 can facilitate the nuclear localization of YAP 35 . DLG5 has a similar structure as ZO2 and contains 4 PDZ domains. In our study, Scribble showed significantly decreased expression in MCF10A-shDLG5 and MCF7-shDLG5 cells and increased expression in MDA-MB-231-oxDLG5 cells (Fig. 5A). Additionally, we observed increased mislocalization of Scribble in MCF10A-shDLG5 cells (Fig. 5B). Interestingly, co-IP assays showed that DLG5 interacted with Scribble (Fig. 5C). Loss of DLG5 expression impaired the interactions of Mst1/2 and Lats1/2 with Scribble. The results suggest that DLG5 might be an upstream regulator of the Hippo signaling pathway.
To ascertain whether DLG5 has an effect on YAP expression and localization, a downstream effector of the Hippo signaling pathway, we examined the expression of the major members of the Hippo signaling pathway in MCF10A-shDLG5 and MCF7-shDLG5 cells. We found that loss of DLG5 expression decreased the levels of p-MST1/2, SAV1, p-MOB1, p-LAST1, p-YAP S127 and p-YAP S397 (Fig. 5D). YAP phosphorylation on Ser127 was also decreased, which resulted in reduced YAP-14-3-3 binding and YAP cytoplasmic retention 36 . A subsequent study revealed apparently increased nuclear YAP and TEAD transcription in MCF10A-shDLG5 and MCF7-shDLG5 cells compared with NC cells (Fig. 5E,F,G). Furthermore, loss of DLG5 expression increased the expression of CTGF and CYR6, two genes downstream of TEAD (Fig. 5H). As predicted, knockdown of YAP in MCF10A-shDLG5 cells decreased TEAD transcriptional activity (Fig. 5I,J).
Interestingly, loss of DLG5 not only increased YAP expression but also decreased p-YAP S397 levels (Fig. 5D). A recent report showed that β -Trcp recognizes YAP phosphorylation on Ser397 and triggers YAP degradation 37 . We detected a significant decrease in β -Trcp expression in MCF10A-shDLG5 cells (Fig. 5K). In contrast, in NC cells, loss of DLG5 promoted YAP stabilization when protein synthesis was not blocked with cycloheximide (CHX). Additionally, YAP accumulated in NC cells, but not in shDLG5 cells, indicating that loss of DLG5 expression might inhibit YAP degradation (Fig. 5L). Taken together, these data demonstrate that loss of DLG5 expression inhibits the Hippo signaling pathway and increases the expression and nuclear localization of YAP.
In vivo studies showed similar results. MCF7-NC or MCF7-shDLG5 cells were subcutaneously injected into nude mice. Knockdown of DLG5 increased tumor weight and volume (Fig. 6A,B,C) and elicited a remarkable increase in YAP and a decrease in Scribble (Fig. 6D,E). Both the in vitro and in vivo results showed that knockdown of DLG5 inactivated the Hippo signaling pathway by decreasing Scribble expression and leading to its mislocalization in the cytoplasm, consequently inducing EMT, loss of cell polarity, cell migration and cell proliferation (Fig. 6F).

Discussion
We are the first to report that loss of DLG5 increased YAP nuclear localization, inhibited YAP degradation, down-regulated Scribble expression, and mislocalized Scribble from the membrane to the cytoplasm; these findings provide insight into the mechanism underlying breast cancer progression. Specifically, loss of DLG5 down-regulated and mislocalized Scribble, inhibited the Hippo signaling pathway by decreasing the interaction of Scribble with Mst1 and Lats1, and increased YAP nuclear localization and TEAD transcription, thereby promoting the proliferation of breast epithelial cells and breast cancer cells, inducing EMT, disrupting the maintenance of cell polarity, and increasing cell migration and invasion.
DLG5 was highly expressed in normal breast tissues/cells and low-grade breast cancer tissues/cells, but its expression was reduced or lost in high-grade breast cancer tissues/cells. Obviously, DLG5 is closely related to breast cancer development. In addition, we showed that DLG5 expression is positively associated with ER/PR status, an important indicator for breast cancer therapy and prognosis. Previous studies have reported that DLG5 is a primary target of progesterone and shows high expression in luminal breast cancer 8,38 . These results suggest that DLG5 may be correlated with cancer therapy and prognosis.
In 1998, Nakamura et al. reported that DLG5 may regulate cell proliferation 39 , but these researches did not ascertain the detailed role of DLG5 in this process. We found that DLG5 expression positively correlated with p53 expression and was negatively associated with Ki67 expression. Both p53 and Ki67 are notable markers of cell proliferation 17 . Interestingly, we also observed that DLG5-knockdown cells grew faster than NC cells. Our results verified the conclusion by Nakamura et al. that DLG5 regulates cell proliferation. p53 has been reported to play a paramount role in inducing G1 and G2/M arrest. Loss of p53 expression causes cells to avoid cell cycle progression checkpoints and induces cell proliferation and tumorigenesis 18 . p21 and p27 are important inhibitors of G1 progression and hallmarks of cell overgrowth 40 . Overexpression of cyclin D1 enables cells to pass the G1/S checkpoint and thus increases the percentage of cells in S phase 41 . According to previous reports, overexpression of cyclin D1 and loss of p53, p21 and p27 expression induce cell overgrowth and are significant markers of malignant cancer 18,42,43 . Our study generated similar results; DLG5 knockdown reduced p53, p21 and p27 expression  DLG5 is involved in regulating the maintenance of epithelial polarity, EMT, and cell migration and invasion 8 . We found that DLG5 co-localized with E-cadherin at cell-cell junctions in the MCF10A breast epithelial cell line. Knockdown of DLG5 disrupted breast cell acinar polarity by inducing the mislocalization of E-cadherin and GM130. Additionally, loss of DLG5 expression induced EMT by altering the localization and expression of EMT markers, specifically by decreasing ZO1 expression and increasing N-cadherin and vimentin expression. However, E-cadherin expression did not change in response to changes in DLG5 expression. Our results suggest that loss of DLG5 might disrupt the maintenance of cell polarity, accompanied by partial EMT.
The Hippo signaling pathway is an evolutionarily conserved regulator of cell proliferation, cell polarity maintenance and tumor suppression. Alterations in this pathway are increasingly recognized to be associated with cancer development 2,5,44 . The mammalian core kinase components comprise MST1/2, SAV1, LATS1/2 and MOB1 34,44 . YAP is a downstream effector of the Hippo pathway, and nuclear YAP is involved in regulating cell proliferation, EMT, and cell polarity maintenance 45 . Phosphorylation of YAP on Serine 127 is crucial for promoting its association with 14-3-3 protein and its subsequent cytoplasmic sequestration and inactivation 3 . YAP phosphorylation on Ser397 is recognized by β -Trcp, an F-box protein that participates in the ubiquitination and degradation of YAP 37 . Previous research showed that DLG5 expression is down-regulated in YAP-overexpressing cells 8 . Recent studies revealed that many cell polarity proteins, such as LKB1 and ZO2, play an important role in regulating the Hippo signaling pathway 35,46 . Thus, we were curious whether DLG5 expression has an effect on the Hippo signaling pathway. We first found that loss of DLG5 expression decreased the levels of p-MST1/2, SAV1, p-MOB1, and p-LAST1, resulting in inactivation of the Hippo signaling pathway. Loss of DLG5 expression decreased p-YAP S127 and p-YAPS 397 levels, thereby inhibiting YAP degradation and increasing YAP nuclear localization.
A recent report showed that Scribble acts downstream of LKB1 to regulate the Hippo signaling pathway 46 . Scribble expression and localization to the plasma membrane are key factors in the activation of the Hippo signaling pathway. Impaired interactions of Mst1 and Lats1 with Scribble inhibit the Hippo signaling pathway and induce the nuclear localization of YAP. We found that loss of DLG5 expression inhibited Scribble expression and induced its mislocalization to the cytoplasm. The interactions of Mst1 and Lats1 with Scribble were decreased in MCF10A-shDLG5 and MCF7-shDLG5 cells. Taken together, loss of DLG5 expression promoted breast cancer malignancy by inactivating the Hippo pathway and increasing nuclear YAP.
In conclusion, loss of DLG5 expression (1) inhibits the Hippo pathway by inducing the mislocalization of Scribble and decreasing its expression; (2) increases YAP nuclear localization; and (3) suppresses p21, p27 and p53 expression. In general, loss of DLG5 expression promotes cell proliferation, EMT, maintenance of cell polarity, and cell migration and invasion by inactivating the Hippo signaling pathway. We expect that our findings may offer a potential therapeutic target for breast cancer.
Ethical approval and consent to participate. All patients signed informed consent forms, and the study was approved by the Ethics Review Committee of the First Affiliated Hospital of Xi'an Jiaotong University College of Medicine. All animal experiments were conducted according to the guidelines of the Institutional Animal Care and Use Committee of the First Affiliated Hospital of Xi'an Jiaotong University.