ROR1 and ROR2 play distinct and opposing roles in endometrial cancer.

OBJECTIVE: In recent years, the Wnt signalling pathway and the ROR1 and ROR2 receptors have been implicated in a range of gynecological cancers. These receptors have been described as prospective therapeutic targets, and this study investigated such potential in an endometrial cancer context. METHOD: Immunohistochemistry for ROR1 and ROR2 was performed in a patient cohort, and expression was correlated with clinicopathological parameters including type, stage, grade, myometrial invasion, lymphovascular involvement, patient age and survival. The functional role of these receptors in endometrial cancer was investigated via siRNA knockdown of ROR1 and ROR2 in three cell line models (KLE, RL95-2 and MFE-319). Effects on proliferation, adhesion, migration and invasion were measured. RESULTS: High ROR1 expression in patient samples correlated with worse overall survival (p = 0.0169) while high ROR2 expression correlated with better overall survival (p = 0.06). ROR1 knockdown in KLE cells significantly decreased proliferation (p = 0.047) and reduced migration and invasion. ROR2 knockdown in RL95-2 cells increased cell migration and invasion (p = 0.011). Double ROR1 and ROR2 knockdown in MFE-319 cells decreased adhesion and significantly increased cell migration (P = 0.008) and invasion (p < 0.001). CONCLUSION: ROR1 and ROR2 play distinct roles in endometrial cancer. ROR1 may promote tumor progression, similar to its role in ovarian cancer, while ROR2 may act as a tumor suppressor in endometrioid endometrial cancer, similar to its role in colorectal cancer. With several ROR-targeting therapies currently in development and phase I clinical trials for other tumor types, this study supports the potential of these receptors as therapeutic targets for women with endometrial cancer.

Migration and invasion is inhibited by silencing ROR1 and ROR2 in chemoresistant ovarian cancer.

Ovarian cancer survival remains poor despite recent advances in our understanding of genetic profiles. Unfortunately, the majority of ovarian cancer patients have recurrent disease after chemotherapy and lack other treatment options. Wnt signalling has been extensively implicated in cancer progression and chemoresistance. Therefore, we investigated the previously described Wnt receptors ROR1 and ROR2 as regulators of epithelial-to-mesenchymal transition (EMT) in a clinically relevant cell line model. The parental A2780- and cisplatin-resistant A2780-cis cell lines were used as a model of ovarian cancer chemoresistance. Proliferation, adhesion, migration and invasion were measured after transient overexpression of ROR1 and ROR2 in the parental A2780 cell line, and silencing of ROR1 and ROR2 in the A2780-cis cell line. Here we show that ROR1 and ROR2 expression is increased in A2780-cis cells, alongside ß-catenin-independent Wnt targets. Knockdown of ROR1 and ROR2 significantly inhibited cell migration and invasion and simultaneous knockdown of ROR1 and ROR2 significantly sensitised cells to cisplatin, whilereas ROR overexpression in the parental cell line increased cell invasion. Therefore, ROR1 and ROR2 have the potential as novel drug targets in metastatic and recurrent ovarian cancer patients.

Wnt signalling in gynaecological cancers: A future target for personalised medicine?

The three major gynaecological cancers, ovarian, uterine and cervical, contribute a significant burden to global cancer mortality, and affect women in both developed and developing countries. However, unlike other cancer types that have seen rapid advances and incorporation of targeted treatments in recent years, personalised medicine is not yet a reality in the treatment of gynaecological cancers. Advances in sequencing technology and international collaborations and initiatives such as The Cancer Genome Atlas are now revealing the molecular basis of these cancers, and highlighting key signalling pathways involved. One pathway which plays a role in all three cancer types, is the Wnt signalling pathway. This complex developmental pathway is altered in most human malignancies, and members of this pathway, particularly the recently linked ROR receptor tyrosine kinases may be attractive future therapeutic targets. This review provides an up-to-date summary of research into Wnt signalling and ovarian, uterine and cervical cancers, and discusses the potential of the Wnt pathway as a future target for personalised medicine in gynaecological cancers.

The C-terminal binding protein (CTBP-1) regulates dorsal SMD axonal morphology in Caenorhabditis elegans.

C-terminal binding proteins (CtBPs) are transcriptional co-repressors which cooperate with a variety of transcription factors to repress gene expression. Caenorhabditis elegans CTBP-1 expression has been observed in the nervous system and hypodermis. In C. elegans, CTBP-1 regulates several processes including Acute Functional Tolerance to ethanol and functions in the nervous system to modulate both lifespan and expression of a lipase gene called lips-7. Incorrect structure and/or function of the nervous system can lead to behavioral changes. Here, we demonstrate reduced exploration behavior in ctbp-1 mutants. Our examination of a subset of neurons involved in regulating locomotion revealed that the axonal morphology of dorsal SMD (SMDD) neurons is altered in ctbp-1 mutants at the fourth larval (L4) stage. Expressing CTBP-1 under the control of the endogenous ctbp-1 promoter rescued both the exploration behavior phenotype and defective SMDD axon structure in ctbp-1 mutants at the L4 stage. Interestingly, the pre-synaptic marker RAB-3 was found to localize to the mispositioned portion of SMDD axons in a ctbp-1 mutant. Further analysis of SMDD axonal morphology at days 1, 3 and 5 of adulthood revealed that the number of ctbp-1 mutants showing an SMDD axonal morphology defect increases in early adulthood and the observed defect appears to be qualitatively more severe. CTBP-1 is prominently expressed in the nervous system with weak expression detected in the hypodermis. Surprisingly, solely expressing CTBP-1a in the nervous system or hypodermis did not restore correct SMDD axonal structure in a ctbp-1 mutant. Our results demonstrate a role for CTBP-1 in exploration behavior and the regulation of SMDD axonal morphology in C. elegans.

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

PURPOSE: Wnt signalling has been implicated in breast cancer, and in particular aberrant ß-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 ß-catenin-dependent and ß-catenin-independent targets and decreased cell adhesion. Knock-down of ROR2 in the breast cancer cell lines, MDA-MB-453 and HCC1143, decreased both ß-catenin-dependent and ß-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 ß-catenin-dependent and ß-catenin-independent Wnt signalling pathways, and represents a potential diagnostic and therapeutic target.

The non-canonical Wnt ligand, Wnt5a, is upregulated and associated with epithelial to mesenchymal transition in epithelial ovarian cancer.

OBJECTIVE: Aberrant Wnt signalling has previously been associated with gynaecological cancers, and the aim of this study was to investigate the expression of Wnt5a in epithelial ovarian cancer, and clarify its role in activating or inhibiting ß-catenin dependent and independent Wnt signalling pathways. METHOD: Wnt5a expression was investigated in a large cohort of epithelial ovarian cancer patient samples using immunohistochemistry and correlated with clinicopathological variables. Wnt5a function was investigated in vitro in ovarian cell lines. RESULTS: Wnt5a expression was found to be upregulated in all major subtypes (serous, endometrioid, clear cell and mucinous) of epithelial ovarian cancer compared to borderline tumours and benign controls. Treatment of ovarian surface epithelial cells with recombinant Wnt5a decreased cell adhesion and was associated with increased epithelial to mesenchymal transition (EMT). In addition, downstream targets of ß-catenin dependent Wnt signalling were inhibited, and ß-catenin independent targets increased following Wnt5a upregulation. Knockdown of Wnt5a in ovarian cancer cells was associated with a mesenchymal to epithelial transition (MET), but had no significant effect on cell migration or proliferation. CONCLUSION: This study adds to the increasing evidence that Wnt signalling may play an important role in ovarian cancer development. Utilising an unparalleled large cohort of 623 patients, Wnt5a protein expression was shown to be significantly higher in ovarian cancer patients when compared to benign and borderline ovarian tumours and healthy control patients. In addition, we have utilised in vitro models to show for the first time in ovarian cancer that Wnt5a driven non-canonical pathways can alter epithelial to mesenchymal transition (EMT).