PLCA8 suppresses breast cancer apoptosis by activating the PI3k/AKT/NF‐κB pathway

Abstract The cysteine‐rich lysosomal protein placenta‐specific 8 (PLAC8), also called onzin, has been shown to be involved in many types of cancers, and its role is highly dependent on cellular and physiological contexts. However, the precise function of PLAC8 in breast cancer (BC) progression remains unclear. In this study, we investigated both the clinical significance and biological functions of PLAC8 in BC progression. First, high PLAC8 expression was observed in primary BC tissues compared with adjacent normal tissues through immunohistochemistry analysis. The results of in vitro and in vivo assays further confirmed that PLAC8 overexpression promotes cell proliferation and suppress BC cell apoptosis, whereas PLAC8 silencing has the opposite effect. In addition, the forced expression of PLAC8 greatly induces cell migration, partially by affecting the EMT‐related genes, including down‐regulating E‐cadherin expression and facilitating vimentin expression. Further mechanistic analysis confirmed that PLAC8 contributes to cell proliferation and suppresses cell apoptosis in BC by activating the PI3K/AKT/NF‐κB pathway. The results of our study provide new insights into an oncogenic role of PLAC8 and reveal a novel PLAC8/ PI3K/AKT/NF‐κB pathway as a potential therapeutic target for BC.

intriguing findings elucidated pivotal oncogenic or tumour suppressor roles for PLAC8 in cancer progression. However, the potential mechanisms and functional role of PLAC8 in BC pathogenesis remain unknown. Apoptosis is considered to be the major mechanism of programmed cell death, and the maintenance of normal physiology and cellular homeostasis occurs through different mechanisms. [11][12][13] Apoptosis regulates diverse cellular processes, including cell proliferation, the cell cycle and survival, 14,15 and the results of previous studies have suggested that the inhibition of PI3K/AKT signalling induces apoptosis. 16,17 In this study, we aimed to determine the expression profile and elucidate the pathological functions of PLAC8 during BC progression, and our results revealed a relationship between endogenous PLAC8 expression and PI3K/AKT pathway activity. PLAC8 knockdown in Bcap-37 cells induced apoptosis (ie cell growth inhibition), and improved cell growth in T47d cells was observed as a result of PLAC8 overexpression. Herein, we demonstrate that PLAC8 acts as a significant factor in BC progression by altering the activity of the PI3k/AKT/NF-κB pathway.

| Tissue specimens
Fifty-five paired BC and adjacent tissues samples were obtained from the Sir Run Run Shaw Hospital (affiliated with Zhejiang University) that had been histopathologically and clinically diagnosed from 2012 to 2018. The tumour grade and metastasis were measured according to the American Joint Committee on Cancer, 8th Edition. All tissues were frozen and stored in liquid nitrogen until analysed. All patients gave informed consent to use excess pathological specimens for research purposes. The study was approved by the Ethics Committee of the Sir Run Run Shaw Hospital, which is affiliated with Zhejiang University.  15 Medium supplemented with 10% foetal bovine serum. All cell lines were cultured at 37°C, and with the exception of MDA-MB-231, they were grown in a humidified atmosphere containing 5% CO 2 . LY294002 was purchased from Selleck (S1105, Texas, USA).

| Transfection
Short interfering RNAs targeting PLAC8 (Si-PLAC8) and a scrambled control siRNA were designed and purchased from RiboBio (Guangzhou, China). The Si-PLAC8 sequences were CCTTGGGTGTCAAGTAFCA (Si-PLAC8#1) and GGAACAAGCGTCGCAATGA (Si-PLAC8#2). Lentiviral PLAC8-overexpressing and negative control vectors were gifts from Dr Yongxia Chen. Cells were transfected with siR-NAs or plasmids in serum-free medium using Lipofectamine 3000 (Invitrogen, USA) according to the manufacturer's instructions. at room temperature. Goat anti-mouse IgG H&L (ab6708) and goat anti-rabbit IgG H&L (ab6721) were purchased from Abcam. Reactive bands were visualized with ECL Plus reagents using a LAS-4000 mini instrument. The total protein band intensities were normalized to the loading control (GAPDH) and qualified using ImageJ.

| Immunohistochemical staining
Slices of paraffin-embedded tissues were deparaffinized and rehydrated in xylene and graded alcohol solutions and then blocked with All images were captured using a fluorescence microscope (Olympus BX-51, Japan).

| Immunofluorescence staining
Cells were briefly seeded onto glass coverslips in 24-well plates until reaching 50%-60% confluence. Cells were washed 3 times and

| Cell proliferation assay
Cells (0.7 × 10 4 ) were seeded into a 96-well culture plate for 12, 24 or 72 hours. Cell viability was evaluated using an MTT assay (CellTiter 961 AQueous One Solution Cell Proliferation Assay, Promega). The absorbance was measured at 490 nm using a BioTek ELx800 absorbance microplate reader.

| Wound-healing assay
Cells (5 × 10 5 ) were seeded into 6-well plates and incubated until reaching 80%-90% confluence. Scratch wounds were made using a pipette tip 48 hours after transfection. Subsequently, the cells were washed with PBS 3 times to remove cell debris and then were incubated in complete medium. The scratch was recorded under a phase-contrast microscope at the time of wound generation (0 hour) and at 24 hours. The gap widths were measured using ImageJ.

| Apoptosis and cell cycle flow cytometry
Cells were seeded into 6-well plates and then collected after

| TUNEL apoptosis assay
TUNEL apoptosis assays were performed with a KeyGEN Biotech kit (KGA7052, Jiangsu, China) according to the manufacturer's protocol.
The cells were seeded into 6-well plates and cultured for 24 hours.
Following transfection for 48 hours, the cells were collected, and the cells or frozen slides of nude mice were blocked with 3% H 2 O 2 in methanol. A sufficient volume of proteinase K was added to completely cover the cells or tissue, and the samples were incubated for 30 minutes in a humidified chamber at room temperature. After washing the slides three times with PBS for 5 minutes, 50 μL of TdT reaction buffer was added to each slide, and the slides were incubated for 1 hour at room temperature in the dark. The cells or slides were subsequently washed with PBS three times for 5 minutes each, after which 50μl of streptavidin-FITC reaction buffer was added to each slide. The cells or slides were then incubated for 30 minutes at room temperature in the dark, and then, images were acquired using a Nikon laser scanning confocal microscope (Nikon Instruments Inc, Melville, NY, USA).

| RNA isolation and quantitative real-time PCR
Total RNA was extracted from cells and tissues using TRIzol reagent

| Tumour xenografts in nude mice
Twelve BALB/c nude mice (aged 4-6 weeks, from Shanghai Laboratory Animal Center, Shanghai, China) were housed in a specific pathogen-free environment. According to the expression of the target genes, nude mice were randomly divided into two groups: Control (with empty vector) or PLAC8 (overexpressing PLAC8) (n = 6). T47D cells (2 × 10 6 ) transfected with a PLAC8-overexpressing lentivirus or empty vector in 100μl of PBS with 100μl of growth factor-reduced basement membrane matrix (Corning Costar, Cambridge, MA, USA) were injected into the right subaxillary region of each mouse.
The tumour size was measured using a slide calliper twice per week, and the tumour volume was calculated using the following formula: (A × B 2 )/2 (A, the length of the tumour; B, the width of the tumour).
Eighteen days after injection, the mice were killed, and the subcutaneous growth of each tumour was examined. The wet tumour weight was calculated as the mean weight ± standard deviation (SD) for each group. This study was approved by the Ethics Committee for Animal Studies of Zhejiang University (Hangzhou, China).

| PLAC8 expression is frequently up-regulated in breast cancer
To date, the results of several studies have strongly suggested that PLAC8 regulates cell division, differentiation and apoptosis. 9,18,19 To determine the biological function of PLAC8 in BC, we first evaluated the level of PLAC8 mRNA in 55 samples of BC and their corresponding adjacent tissues. The level of PLAC8 mRNA was increased in BC tumour tissues ( Figure 1A). Furthermore, we analysed the IHC staining results for the samples and showed that PLAC8 was up-regulated in the cancer tissues compared with the adjacent tissues ( Figure 1B). In addition, PLAC8 was observed to be closely correlated with tumour size and TNM stage ( Table 1, Table S1). These findings suggest that PLAC8 may contribute to BC progression. In addition, we assessed the expression of PLAC8 protein in various BC cell lines and observed that PLAC8 expression was relatively higher in Bcap-37 and MDA-MB-231 cells than in the other cells assayed ( Figure 1C).
Unsurprisingly, the expression of PLAC8 was dramatically different in various BC cell lines. We further confirmed the subcellular location of PLAC8 using immunofluorescence staining with a PLAC8-specific antibody, the results of which showed that PLAC8 was primarily labelled in the cytoplasm ( Figure 1D). Taken together, these data strongly indicate that PLAC8 is associated with BC progression.
F I G U R E 2 PLAC8 promotes cell proliferation in vitro. A and B, Bcap-37 and T47D cells were transfected with PLAC8 siRNA and/or the PLAC8 overexpression plasmid, and the interference effect of siRNA or plasmid was determined by Western blotting (upper) 72 h after transfection and qRT-PCR (bottom) 48 h after transfection. The expression was quantified and normalized to GAPDH. The error bars correspond to the means ± SD. (C) Cell proliferation assays were performed to determine the cell viability for Bcap-37 and T47D cells transfected with PLAC8 siRNA or plasmid. The error bars correspond to the means ± SD. The values are presented as the means ± SD * P < 0.05; ** P < 0.01; NS, not significant and Si-PLAC8#2) or non-targeting control (Si-NC) and T47D cells transfected with the PLAC8 or vector plasmids (Figure 2A,B). As shown in Figure 2A

| PLAC8 silencing induces caspase 3/9 activation, Bcl-2 up-regulation and apoptosis of breast cancer cells
Recent studies have shown that the aberrant regulation of apoptosis results in uncontrolled cell proliferation. [20][21][22] As PLAC8 overexpression facilitates BC cell proliferation, we assessed whether increased or decreased PLAC8 expression contributes to cell apoptosis to influence BC progression. The results revealed that PLAC8transfected T47D cells were inhibited for apoptosis, whereas PLAC8 knockdown significantly induced apoptosis in Bcap-37 cells ( Figure 4A,B). Furthermore, we determined the expression of apoptosis-related markers by Western blotting ( Figure 5A).

| PLAC8 suppresses cell apoptosis via the PI3k/ AKT/NF-κB pathway
We speculated that PLAC8 regulates BC cell apoptosis in vitro and in vivo by regulating the expression of different target genes. To further elucidate the mechanisms by which PLAC8 inhibits BC cell apoptosis, we examined the putative upstream regulators and downstream targets of PLAC8. The PI3K/AKT pathway not only plays a central role in cell cycle distribution, survival and drug sensitivity but also is associated with cell growth and apoptosis. 17,[27][28][29] Because these properties make the PI3K/AKT pathway a major candidate for investigating cell proliferation, we aimed to examine the potential relationship between PLAC8 and the PI3K/AKT pathway in BC. To determine whether PLAC8 regulates apoptosis via this pathway in BC cells, the levels of PI3K/AKT pathway-associated molecules and their phosphorylated forms were examined. The results of Western blot analyses showed that the levels of PI3K p85, pPI3K, AKT, pAKT-s473 and pNF-κB p65 were significantly higher in T47D cells overexpressing PLAC8 ( Figure 7A). In addition, changes in the expression of PI3K p85, pPI3K, AKT and pAKT-s473 in response to PLAC8 significantly were attenuated by LY294002, a strong inhibitor of phosphoinositide 3-kinases (PI3Ks) ( Figure 7B). Taken together, these results F I G U R E 4 PLAC8 inhibits apoptosis both in vivo and vitro. A and B, Bcap-37 and T47D cells were stained with Annexin V/PI or Tunnel solution, and the percentage of apoptotic cells was determined using a flow cytometer 48 h after transfection. An enlarged view of the boxed area from each group confirms the presence of TUNEL-positive cells. The values are presented as the means ± SD ** P < 0.01; NS, not significant indicate that PLAC8 regulates the apoptosis of BC cells through the PI3K/AKT/NF-κB pathway.

| D ISCUSS I ON
The results of our study provide direct evidence for the pro-tumori- However, in hepatocellular carcinoma, PLAC8 is a tumour suppressor that is regulated by miR-185-5p, which suppresses cell proliferation. 32 The precise role and underlying mechanism of action of PLAC8 in various cancers, especially BC, remain unclear, and our results further our understanding of the cellular functions of this protein in BC.
In this study, we observed that the inhibition of PLAC8 expres- In summary, in this study, we showed that PLAC8 is an oncogene that can actively promote cell viability and proliferation in vitro and in vivo by acting as an upstream regulator that activates the PI3K/ Akt/NF-κB pathway in BC. Taken together, these results reveal a F I G U R E 7 PLAC8 knockdown induces apoptosis and affects cell proliferation by inhibiting the PI3K/AKT/NF-κB pathway. A, Western blot analysis of Bcap-37 or T47D cells 72 h after PLAC8 siRNA or plasmid transfection. Scramble RNA or vector plasmid was used as negative controls. The levels of PI3K/ AKT/NF-κB pathway-related markers were measured. Protein expression was quantified and normalized to GAPDH. The error bars correspond to the means ± SD. (B) Cells were transfected for 48 h and subsequently cultured with or without LY294002 (10 μm) for 24 h. Western blot analysis showing changes in the levels of PI3K pathway-related markers. Protein expression was quantified and normalized to GAPDH. The error bars correspond to the means ± SD. The values are presented as the means ± SD * P < 0.05; ** P < 0.01; NS, not significant novel role for PLAC8 in tumorigenesis and provide novel insights into the molecular pathogenesis of BC.

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