Expression of the novel Wnt receptor ROR2 is increased in breast cancer and may regulate both β-catenin dependent and independent Wnt signalling

Abstract

Purpose: Wnt signalling has been implicated in breast cancer, and in particular aberrant β-catenin-independent Wnt signalling has been associated with breast cancer metastasis and Tamoxifen resistance. Despite Wnt pathway involvement in many human cancers, attempts to target the pathway therapeutically have been disappointing. The recent discovery that the receptor tyrosine kinase-like orphan receptor 2 (ROR2) is a novel Wnt receptor provides a potential new therapeutic and diagnostic target.

Methods: To clarify the role of ROR2 in breast cancer, we investigated its expression via ROR2 immunohistochemistry in a clinical cohort of breast cancer patients, and via in vitro studies incorporating both overexpression and knock-down of ROR2.

Results: ROR2 was expressed in the majority of breast cancer patients (87%), including those classed as triple negative. Breast cancer patients expressing ROR2 had a significantly shorter overall survival than those lacking ROR2 expression (P < 0.05). Overexpression of ROR2 in the mammary epithelial cell line, MCF10A, increased both β-catenin-dependent and β-catenin-independent targets and decreased cell adhesion. Knock-down of ROR2 in the breast cancer cell lines, MDA-MB-453 and HCC1143, decreased both β-catenin-dependent and β-catenin-independent targets and increased cell adhesion. Treatment of ROR2-expressing breast cancer cells with the novel berberine derivative, NAX53, significantly inhibited cell proliferation and migration.

Conclusions: This is the first study to report the expression of ROR2 in breast cancer. Breast cancer patients expressing ROR2 had a significantly worse prognosis than those lacking ROR2. ROR2 may regulate both β-catenin-dependent and β-catenin-independent Wnt signalling pathways, and represents a potential diagnostic and therapeutic target.

Fig. 1

ROR2 protein expression is increased in breast cancer patients and linked to disease-specific survival. a Examples of 0, 1, 2, or 3+ cytoplasmic ROR2 IHC in breast cancer cells. Immunoperoxidase with DAB chromogen and haematoxylin counterstaining. b Breast cancer patients with ROR2 expression (n = 203) had a statistically shorter (P = 0.02) disease-specific survival compared to patients lacking ROR2 expression (n = 31). c Disease-specific survival stratified by ROR2 IHC score. d Triple-negative breast cancer (TNBC) patients (n = 53) had a shorter disease-specific survival than non-TNBC patients (n = 249). e Patients classified as both TNBC and ROR2 positive (n = 37) had the worst prognosis of all patient groups, when compared to TNBC−/ROR2− (n = 26), TNBC+/ROR2− (n = 5), or TNBC−/ROR2+ (n = 161)

Fig. 2

ROR2 upregulates β-catenin-independent Wnt signalling in mammary epithelial cells. a Densitometric analysis of ROR2 protein expression in three separate experiments. Error bars represent the standard deviation (SD) of the mean. *P < 0.05. b Representative immunoblot showing ROR2 is increased at the protein level following transfection with a ROR2-FLAG plasmid in MCF10A mammary epithelial cells. Top panel ROR2, bottom panel α-tubulin. c Densitometric analysis of MYC and JUN protein expression in three separate experiments. Error bars represent the SD of the mean. d Representative immunoblots showing β-catenin-dependent and β-catenin-independent Wnt target genes are increased at the protein level following transfection with a ROR2-FLAG plasmid in MCF10A mammary epithelial cells. Top panel JUN, middle panel MYC, bottom panel α-tubulin. In all graphs, black bars represent the empty vector control, dark grey bars represent ROR2-FLAG plasmid A, light grey bars represent ROR2-FLAG plasmid B

Fig. 3

ROR2 decreases adhesion and is associated with EMT in mammary epithelial cells. a ROR2 transfected MCF10A cells did not proliferate significantly faster than control cells. Three independent experiments were conducted, and the results averaged. b ROR2-transfected MCF10A cells migrated slightly slower than control cells at 24 h, though this may be due to the minor differences in proliferation. The graphs represent the average of three independent experiments. c ROR2 decreased adhesion to collagen and fibronectin. Results represent the average of three experiments and bars represent the standard deviation (SD) of the mean. *P < 0.05. d Densitometric analysis of VIM and CDH1 protein expression in three separate experiments. Error bars represent the (SD) of the mean. e Representative immunoblots showing VIM is increased, and CDH1 decreased at the protein level following transfection with a ROR2-FLAG plasmid in MCF10A mammary epithelial cells. Top panel VIM, middle panel CDH1, bottom panel α-tubulin. In all graphs, black bars represent the empty vector control, dark grey bars represent ROR2-FLAG plasmid A, light grey bars represent ROR2-FLAG plasmid B

Fig. 4

Knock-down of ROR2 in TNBC cells inhibits both canonical and non-canonical Wnt signalling. a ROR2 is decreased at the mRNA level following siRNA-induced knock-down in HCC1143 TNBC. qRT-PCR was performed in triplicate and normalised to three different housekeeping genes (SDHA, HSPCB, RPL13A). Results represent an average of six experiments. Error bars represent the SD of the mean. *P < 0.05. b Densitometric analysis of ROR2 protein levels from five separate experiments. c Representative immunoblots showing ROR2 knock-down at the protein level in HCC1143 cells. Top panel ROR2, bottom panel α-tubulin. d β-Catenin-independent target genes are unaffected at the mRNA level following ROR2 knock-down. qRT-PCR was performed in triplicate and normalised to three different housekeeping genes (SDHA, HSPCB, RPL13A). Results represent an average of three experiments. Error bars represent the SD of the mean. e β-Catenin-dependent target genes are altered following ROR2 knock-down. qRT-PCR was performed in triplicate and normalised to three different housekeeping genes (SDHA, HSPCB, RPL13A). Results represent an average of three experiments. Error bars represent the SD of the mean. *P < 0.05. f Densitometric analysis of three separate experiments indicating that both β-catenin-dependent and β-catenin-independent target genes are decreased at the protein level following ROR2 knock-down. Error bars represent the SD of the mean. *P < 0.05. g Representative immunoblots of Wnt target genes following ROR2 knock-down. Top panel RHOA, second panel JUN, third panel WNT5A, fourth panel MYC, fifth panel CCND1, bottom panel α-tubulin. h TCF/LEF Wnt reporter assay. Results are the average of two independent experiments. In all graphs, black bars represent the non-targeting control siRNA, and grey bars represent ROR2 siRNA knock-down

Fig. 5

Knock-down of ROR2 in TNBC cells alters cell behaviour. a Knock-down of ROR2 in HCC1143 cells does not significantly alter cell proliferation over a 24-h period. Results represent the average of three independent experiments. b Cell migration is increased following ROR2 knock-down in HCC1143 cells (n = 3). c Cell adhesion is slightly increased following ROR2 knock-down in HCC1143 cells (n = 3). d EMT markers do not change significantly at the mRNA level following knock-down in HCC1143 cells. (qRT-PCR was performed in triplicate and normalised to three different housekeeping genes (SDHA, HSPCB, RPL13A). Results represent an average of three experiments. Error bars represent the SD of the mean. e Densitometric analysis of three separate experiments indicates that CDH1 is increased and VIM decreased at the protein level following knock-down in HCC1143 cells. f Representative immunoblot indicating CDH1 is increased and VIM decreased at the protein level following knock-down in HCC1143 cells. Top panel CDH1, middle panel VIM, bottom panel α-tubulin. In all graphs, black bars represent the non-targeting control siRNA, and grey bars represent ROR2 siRNA knock-down

Fig. 6

Treatment of TNBC cells with drugs targeting the Wnt pathway inhibits cell proliferation, migration and EMT. a Densitometric analysis of three separate experiments indicates Wnt pathway and EMT components are altered at the protein level following treatment with NAX 53. b Representative immunoblots of Wnt and EMT targets altered following NAX53 treatment of HCC1143 cells. c NAX53 treatment significantly decreases cell proliferation over 24 h (n = 3). *P < 0.05. d NAX53 treatment significantly inhibits cell migration over 24 h (n = 3). **P < 0.01