ROR2 promotes invasion and chemoresistance of triple-negative breast cancer cells by activating PI3K/AKT/mTOR signaling

Objective This study aimed to investigate the role of receptor tyrosine kinase-like orphan receptor 2 (ROR2) in triple-negative breast cancer (TNBC). Methods ROR2 expression in primary TNBC and metastatic TNBC tissues was analyzed by immunohistochemical staining and PCR. ROR2 expression in TNBC cell lines was detected by PCR and Western blot analysis. The migration, invasion and chemosensitivity of TNBC cells with overexpression or knockdown of ROR2 were examined. Results ROR2 expression was high in metastatic TNBC tissues. ROR2 knockdown suppressed the migration, invasion and chemoresistance of TNBC cells. ROR2 overexpression in MDA-MB-435 cells promoted the migration, invasion, and chemoresistance. Moreover, ROR2 knockdown in HC1599 and MDA-MB-435 adriamycin-resistant cells enhanced chemosensitivity to adriamycin. ROR2 could activate PI3K/AKT/mTOR signaling in TNBC cells. Conclusion ROR2 is upregulated and promotes metastatic phenotypes of TNBC by activating PI3K/AKT/mTOR signaling.


Introduction
Breast cancer is a common cancer in the women [1,2].Triplenegative breast cancer (TNBC) is named due to negative expression of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2) [3,4].Although treatment can extend the survival of patients, one in five patients with TNBC will develop chemoresistance and metastatic disease within five years after the diagnosis [5].TNBC has the worst outcome among breast cancer subtypes, with five-year survival rate of 91%, 65%, 11% for localized, regional and metastatic TNBC, respectively [6].
Therefore, this study aimed to analyze the expression of ROR2 in metastatic TNBC and investigate whether ROR2 regulates PI3K/AKT/mTOR signaling in metastatic TNBC.

Immunohistochemistry
The study was approved by Ethics Committee of Nanjing First Hospital and all patients provided informed consent.Total 40 breast cancer specimens were used, including 20 from patients with TNBC primary cancer and 20 from patients with TNBC metastatic cancer (metastasized to the brain).The tissues were cut into 4 µm sections after fixing in formalin and embedding in paraffin.After incubation with ROR2 antibody (EnoGene), the staining was detected by DAB kit (Beyotime).

CCK-8 assay
Cell viability was examined by CCK-8 assay (EnoGene, New York, USA).Approximately 1 × 10 4 TNBC cells were seeded in each well of 96-well plates.After culture, 10 μL of CCK-8 solution was added and incubated for 4 h, and absorbance at 450 nm was measured by microplate reader (Thermo Fisher, Waltham, MA, USA).

Wound healing assay
Cells were seeded in 6-well plates (Corning, Corning, NY, USA).When cells reached 90% confluency, cell monolayers were scratched with tips.Wound healing was monitored at 0 and 24 h after scratch, and the images were captured under a light microscope with 10× objective.The wound area (cell-free area) was measured by using a digital camera fitted to the microscope.The wound area in control group was set as 1 to calculate relative wound area in experimental groups.

Cell invasion assay
About 1 × 10 5 cells were seeded in serum-free medium on the upper surface of modified Boyden chambers (Millipore, USA) and the lower chamber was loaded with 10% FBS.After incubation for 24 h, the invading cells were stained with 0.5% violet dye and counted under a light microscope with 20× objective in five randomly selected fields.

Flow cytometry
Cell apoptosis was detected using apoptosis detection kit (Enjing, China).Briefly, the cells (5 × 10 5 ) were washed and resuspended in 500 μL binding buffer.The cells were then incubated with 5 μL Annexin V-FITC and 5 μL propidium iodide on the ice in the dark.Fluorescence of 1 × 104 cells per sample was analyzed on a flow cytometer (BD Biosciences, UK).

Statistical analysis
The data are presented as mean ± standard deviation (SD).Comparisons for multiple groups were performed by oneway analysis of variance, with significance set as p < 0.05.

ROR2 expression was positively correlated with TNBC metastasis
First, we examined ROR2 expression in primary and metastatic TNBC tissues by IHC staining and PCR.Strong ROR2 staining was observed in the invasive and metastatic TNBC tissues (Figs.1A and 1B).In TNBC tumors with metastasis, ROR2 expression was higher than that in metastasis-negative tumors (Fig. 1C).Furthermore, we analyzed ROR2 mRNA and protein levels in TNBC cells.Among them, ROR2 expression was the highest in HCC1599 cells and the lowest in MDA-MB-435 cells (Figs. 1D-1F).We selected HCC1599 and MDA-MB-435 cells for ROR2 knockdown and overexpression, respectively.
ROR2 activated PI3K/AKT/mTOR signaling in TNBC cells Western blot analysis showed that the levels of p-PI3K, p-AKT and p-mTOR increased in ROR2 overexpressing MDA-MB-435 cells.However, ROR2 knockdown in HCC1599 cells reduced the levels of p-PI3K, p-AKT, and p-mTOR with no significant changes in PI3K, AKT, and mTOR levels (Fig. 4A).Furthermore, p-AKT, p-PI3K and p-mTOR levels were significantly higher in tissues from metastatic TNBC compared to those in tissues from primary TNBC cancer (Fig. 4B).
Furthermore, ROR2 overexpressing cells showed the upregulation of N-cadherin, vimentin, MMP-2 and Snail,   and the downregulation of E-cadherin, compared to MDA-MB-435 parental cells.The expression of these proteins in HCC1599/siRNA-ROR2 cells showed the opposite trend compared to HCC1599 parental cells (Fig. 6).

Discussion
ROR2 has been proposed as an oncogene [13,14].Moreover, ROR2 expression was increased in NSCLC [15].In this study, we showed that higher ROR2 expression in TNBC tissues correlated with metastatic phenotype in TNBC, indicating that ROR2 may promote TNBC metastasis.
PI3K/AKT/mTOR pathway is often deregulated in different cancers [18][19][20][21].Previous studies have shown that certain small molecule compounds exert inhibitory effects on breast cancer by inhibiting PI3K/AKT signaling [22].Rehman et al. demonstrated that downregulation of ROR2 inhibited thyroid cancer cell proliferation and invasion via PI3K/AKT signaling [23].Here, we showed that ROR2 knockdown inhibited TNBC cell migration and invasion and increased chemosensitivity in vitro.
Recognized as a noncanonical Wnt receptor, ROR2 regulates a variety of cell activities such as breast cancer cell invasion [24].Regulation of ROR2 by siRNA mediated knockdown may inhibit all downstream signaling pathways, including but not limited to PI3K/AKT/mTOR signaling.In contrast, small molecule compounds of PI3K/AKT signaling inhibitors are specific and exclusive.
This study has limitations.The sample size is relatively small and studies with large sample size are necessary to confirm high expression of ROR2 in metastatic TNBC tissues.
In conclusion, for the first time we demonstrate that ROR2 and PI3K/AKT/mTOR signaling regulate drug resistance and metastasis in TNBC.ROR2 may be a novel target to limit TNBC invasion and metastasis.

FIGURE 2 .
FIGURE 2. Generating HCC1599 and MDA-MB-435 cells with stable knockdown and overexpression of ROR2.HCC1599 cells were transfected with ROR2 siRNA plasmid or non-targeting control, and selected by 5 μg/ml puromycin.Stable ROR2 expression MDA-MB-435 cells were generated by transfection with pLenti-ROR2 plasmid and selected by 5 μg/ml puromycin.ROR2 expression was analyzed at mRNA (A) and protein (B and C) levels.Data are presented as mean ± SD (n = 3, **p < 0.01 vs. parent or vector transfected control cells).

FIGURE 3 .
FIGURE 3. ROR2 regulated Adriamycin induced apoptosis of TNBC cells.MDA-MB-435 (A and B) and HCC1599 (A and C) cells with altered ROR2 expression were exposed to 2 uM adriamycin and apoptosis was examined by flow cytometry.ROR2 altered chemosensitivity of MDA-MB-435 (D and E) and HCC1599 (D and F).CCK-8 assay of cells treated with adriamycin for 72 h, and IC50 values were calculated (D and E).Data are presented as mean ± SD (n = 3, **p < 0.01 vs. parent or vector control cells).

FIGURE 5 .
FIGURE 5. ROR2 promoted migration and invasion potential of TNBC cells.(A) Representative images (Left panel) showing wound closure after 24 h in TNBC cells.magnification 200×.Right panel: The quantification of wound area.Cells that invaded to the lower side of the chamber were counted (B).Transwell invasion assay.(C and D) The quantification of invaded cells.Scale bar: 100 μm.Data are presented as mean ± SD (n = 5, **p < 0.01 vs. controls).