ROR1 and ROR2 expression in pancreatic cancer.

BACKGROUND: The Wnt receptors ROR1 and ROR2 are generating increased interest as cancer therapeutic targets but remain understudied in pancreatic ductal adenocarcinoma (PDAC). Compared to canonical Wnt/ ß-catenin signalling, the role of noncanonical Wnt signalling in PDAC remains largely unknown. Only one study has investigated the prognostic significance of the noncanonical Wnt signalling receptor, ROR2 in PDAC. No studies have investigated the prognostic role of ROR1 in PDAC. METHODS: Here, we performed analysis of ROR1 and ROR2 mRNA expression in three publicly available datasets ICGC-PACA-AU (n = 81), TCGA-PAAD (n = 150) and CPTAC-PDAC (n = 137). ROR1 and ROR2 protein expression from the CPTAC-PDAC discovery cohort were also analysed. Immunohistochemistry (IHC) using the validated anti ROR1 monoclonal antibody (4A5) was performed on the Australian Pancreatic Cancer Genome Initiative (APGI) cohort of PDAC samples (n = 152). Association between ROR1 cytoplasmic staining intensity and clinicopathological parameters including stage, grade and overall survival (OS) was investigated. RESULTS: High ROR1 mRNA expression levels correlated with a favourable OS outcome in all of the ICGC-PACA-AU, TCGA-PAAD and CPTAC-PDAC cohorts. ROR1 protein expression was not associated with stage, grade or OS in the APGI cohort. CONCLUSION: ROR1 and ROR2 have potential as prognostic markers when measured at the mRNA level in PDAC. Our IHC cohort did not support ROR1 protein expression in predicting OS, and highlighted the discrepancy of prognostic biomarkers when measured by MS, IHC and RNAseq.

Expression of Ror2 Associated with Fibrosis of the Submandibular Gland.

The submandibular gland (SMG) is one of the major salivary glands that play important roles for variety of physiological functions, such as digestion of foods, prevention of infection, and lubrication of the mouth. Dysfunction of the SMG, often associated with a salivary inflammation, adversely influences a person's quality of life. However, the mechanism underlying inflammation-driven dysfunction of the SMG is largely unknown. Here, we used a mouse model in which the main excretory duct of the SMG is ligated unilaterally to induce inflammation of the gland and examined the expression of Wnt5a, Ror1 and Ror2 genes, encoding Wnt5a ligand and its cognate receptors, which have been implicated in tissue damage or inflammatory responses in variety of tissues. We show that expression levels of Ror1, Ror2, and Wnt5a are increased in the ligated SMG undergoing interstitial fibrosis, which is accompanied by robust expression of fibrosis-associated genes, such as TGF-ß1, TNF-α, IL-1ß, and MMP-2. Increased immunostaining signal of Ror2 was detected in the fibrotic tissues with abundant accumulation of fibroblasts and collagen fibers in the ligated SMG, suggesting that Ror2-mediated signaling might be activated in response to tissue damage and associated with progression of fibrosis in the SMG.Key words: submandibular gland, Ror2, Wnt5a, fibrosis, inflammation.

Comparative study of ROR2 and WNT5a expression in squamous/adenosquamous carcinoma and adenocarcinoma of the gallbladder.

AIM: To investigate the expression and clinical pathological significance of ROR2 and WNT5a in gallbladder squamous/adenosquamous carcinoma (SC/ASC) and adenocarcinoma (AC). METHODS: EnVision immunohistochemistry was used to stain for ROR2 and WNT5a in 46 SC/ASC patients and 80 AC patients. RESULTS: Poorly differentiated AC among AC patients aged > 45 years were significantly more frequent compared with SC/ASC patients, while tumors with a maximal diameter > 3 cm in the SC/ASC group were significantly more frequent compared with the AC group. Positive ROR2 and WNT5a expression was significantly lower in SC/ASC or AC with a maximal mass diameter ≤ 3 cm, a TNM stage of I + II, no lymph node metastasis, no surrounding invasion, and radical resection than in patients with a maximal mass diameter > 3 cm, TNM stage IV, lymph node metastasis, surrounding invasion, and no resection. Positive ROR2 expression in patients with highly differentiated SC/ASC was significantly lower than in patients with poorly differentiated SC/ASC. Positive ROR2 and WNT5a expression levels in highly differentiated AC were significantly lower than in poorly differentiated AC. Kaplan-Meier survival analysis showed that differentiation degree, maximal mass diameter, TNM stage, lymph node metastasis, surrounding invasion, surgical procedure and the ROR2 and WNT5a expression levels were closely related to average survival of SC/ASC or AC. The survival of SC/ASC or AC patients with positive expression of ROR2 and WNT5a was significantly shorter than that of patients with negative expression results. Cox multivariate analysis revealed that poor differentiation, a maximal diameter of the mass ≥ 3 cm, TNM stage III or IV, lymph node metastasis, surrounding invasion, unresected surgery and positive ROR2 or WNT5a expression in the SC/ASC or AC patients were negatively correlated with the postoperative survival rate and positively correlated with mortality, which are risk factors and independent prognostic predictors. CONCLUSION: SC/ASC or AC patients with positive ROR2 or WNT5a expression generally have a poor prognosis.

Receptor tyrosine kinase gene expression profiles of Ewing sarcomas reveal ROR1 as a potential therapeutic target in metastatic disease.

Receptor tyrosine kinases (RTKs) have provided molecular targets for the development of novel, prognosis-improving agents in many cancers; however, resistances to these therapies occur. On the cellular level, one resistance mechanism is attributed to functional RTK redundancies and compensatory cross-signaling, leading to perception of RTKs as signaling and target networks. To provide a basis for better exploitation of this network in Ewing sarcoma, we generated comprehensive qPCR gene expression profiles of RTKs in Ewing sarcoma cell lines and 21 untreated primary tumors. Key findings confirm broad-spectrum RTK expressions with potential for signaling redundancy. Profile analyses with regard to patient risk-group further revealed several individual RTKs of interest. Among them, VEGFR3 and TIE1 showed high-level expressions and also were suggestive of poor prognosis in localized tumors; underscoring the relevance of angiogenic signaling pathways and tumor-stroma interactions in Ewing sarcoma. Of note, compared to localized disease, tumors derived from metastatic disease were marked by global high-level RTK expressions. Nine individual RTKs were significantly over-expressed, suggesting contributions to molecular mechanisms of metastasis. Of these, ROR1 is being pursued as therapeutic target in leukemias and carcinomas, but un-characterized in sarcomas. We demonstrate expression of ROR1 and its putative ligand Wnt5a in Ewing sarcomas, and of an active ROR1 protein variant in cell lines. ROR1 silencing impaired cell migration in vitro. Therefore, ROR1 calls for further evaluation as a therapeutic target in metastatic Ewing sarcoma; and described as a pseudo-kinase with several isoforms, underlines these additional complexities arising in our understanding of RTK signaling networks.

Wnt5a, Ryk and Ror2 expression in glioblastoma subgroups.

BACKGROUND: Wnt5a, a non-canonical Wnt ligand, has been shown to play tumor-promoting or tumor-suppressive roles in different neoplasms. Increased Wnt5a expression and Wnt5a-dependent invasive activity that is mediated by one of its receptors, Ryk, have been reported in glioblastomas. METHODS: We investigated the protein expression of Wnt5a, its receptors Ryk and Ror2, and the canonical Wnt pathway marker ß-catenin in 186 cases of glioblastoma and its variants. Associations with clinicopathological and molecular variables and prognosis were analyzed. RESULTS: All glioblastoma cases expressed Wnt5a, Ryk and Ror2 with a different grade. The expression of both Ryk and Ror2 correlated with that of Wnt5a in glioblastomas. The expression of ß-catenin did not correlate with any of Wnt5a, Ryk or Ror2. Wnt5a expression was significantly different among subgroups of the glioblastoma. However, none of Wnt5a, Ryk or Ror2 had a prognostic impact on glioblastoma. For ß-catenin, a shorter progression-free survival was noted in the glioblastoma with oligodendroglioma component (GBMO) subgroup. CONCLUSIONS: Our results corroborated previous findings of Ryk-mediated Wnt5a effect, and suggested a role for Ror2 in the Wnt5a machinery in glioblastoma.

Wnt5a/Ror2 mediates temporomandibular joint subchondral bone remodeling.

Increased subchondral trabecular bone turnover due to imbalanced bone-resorbing and bone-forming activities is a hallmark of osteoarthritis (OA). Wnt5a/Ror2 signaling, which can derive from bone marrow stromal cells (BMSCs), takes a role in modulating osteoblast and osteoclast formation. We showed previously that experimentally unilateral anterior crossbites (UACs) elicited OA-like lesions in mice temporomandibular joints (TMJs), displaying as subchondral trabecular bone loss. Herein, we tested the role of BMSC-derived Wnt5a/Ror2 signaling in regulating osteoclast precursor migration and differentiation in this process. The data confirmed the decreased bone mass, increased tartrate-resistant acid phosphatase (TRAP)-positive cell number, and enhanced osteoclast activity in TMJ subchondral trabecular bone of UAC-treated rats. Interestingly, the osteoblast activity in the tissue of TMJ subchondral trabecular bone of these UAC-treated rats was also enhanced, displaying as upregulated expressions of osteoblast markers and increased proliferation, migration, and differentiation capabilities of the locally isolated BMSCs. These BMSCs showed an increased CXCL12 protein expression level and upregulated messenger RNA expressions of Rankl, Wnt5a, and Ror2. Ex vivo data showed that their capacities of inducing migration and differentiation of osteoclast precursors were enhanced, and these enhanced capabilities were restrained after blocking their Ror2 signaling using small interfering RNA (siRNA) assays. Reducing Ror2 expression in the BMSC cell line by siRNA or blocking the downstream signalings with specific inhibitors also demonstrated a suppression of the capacity of the BMSC cell line to promote Wnt5a-dependent migration (including SP600125 and cyclosporine A) and differentiation (cyclosporine A only) of osteoclast precursors. These findings support the idea that Wnt5a/Ror2 signaling in TMJ subchondral BMSCs enhanced by UAC promoted BMSCs to increase Cxcl12 and Rankl expression, in which JNK and/or Ca(2+)/NFAT pathways were involved and therefore were engaged in enhancing the migration and differentiation of osteoclast precursors, leading to increased osteoclast activity and an overall TMJ subchondral trabecular bone loss in the UAC-treated rats.

Expression of Ror2 mediates invasive phenotypes in renal cell carcinoma.

Ror2 is a Wnt ligand receptor that is overexpressed in a variety of tumors including clear cell renal cell carcinoma (ccRCC). Here we demonstrate that expression of wild type Ror2 results in increased tumorigenic properties in in vitro cell culture and in vivo xenograft models. In addition, Ror2 expression produced positive changes in both cell migration and invasion, which were dependent on matrix metalloprotease 2 (MMP2) activity. Mutations in key regions of the kinase domain of Ror2 resulted in the abrogation of increased tumor growth, cell migration, and cell invasion observed with expression of wild-type Ror2. Finally, we examined Ror2 expression as a prognostic biomarker for ccRCC utilizing the TCGA ccRCC dataset. High expression of Ror2 showed a significant correlation with higher clinical stage, nuclear grade, and tumor stage. Furthermore, high expression of Ror2 in ccRCC patients correlated with significant lower overall survival, cancer specific survival, and recurrence free survival. Together, these findings suggest that Ror2 plays a central role in influencing the ccRCC phenotype, and can be considered as a negative prognostic biomarker and potential therapeutic target in this cancer.

ROR2 and Wnt5a expression in stage 1 pure testicular seminomas.

OBJECTIVE: To elucidate the prognostic utility of several biomarkers including ROR2/Wnt5a in stage 1 pure seminoma, which is a highly curable tumor in need of prognostic markers to avoid unnecessary treatment. STUDY DESIGN: A total of 47 patients of stage 1 seminoma who underwent radical orchiectomy were included in the study. Tissue microarray-based immunohistochemical analysis of placental alkaline phosphatase, D2-40, c-Kit, Oct-3/4, ROR2, Wnt5a, beta-catenin, CD30, vimentin, pancytokeratin, beta-hCG and p53 was conducted, and relevant clinicopathologic features were assessed. RESULTS: ROR2 protein revealed strong diffuse membranous immunoreactivity (IR) in 12.8% and partial weak IR in 40.4%, respectively. Cytoplasimc Wnt5a IR was observed in 27.7%. ROR2 IR was correlated with Wnt5a IR (p = 0.029) and Oct3/4 IR (p = 0.035). c-Kit IR was correlated with Wnt5a IR (p = 0.034). No significant differences were found between ROR2/Wnt5a protein expression and the prognostically relevant features such as lymphatic invasion or pathologic T stage. Pathologic T stage was not correlated with rete invasion (p= 0.23). The expression or loss of other aforementioned antibodies was not associated with the prognostic clinicopathologic characteristics. CONCLUSION: Our results do not support the relevance of ROR2/Wnt5a as biomarkers in stage 1 pure seminomas. The utility of the explored biomarkers as prognostic or differentiation indicators remains to be clarified.

Heat shock protein-90 inhibitor, NVP-AUY922, is effective in combination with fludarabine against chronic lymphocytic leukemia cells cultured on CD40L-stromal layer and inhibits their activated/proliferative phenotype.

Chronic lymphocytic leukemia (CLL) involves disease infiltration into active proliferation centers within the lymph nodes and marrow. Successful treatment of CLL must involve targeting the leukemic cells in these supportive microenvironments. Our recent data suggest that inhibition of heat shock protein-90 (Hsp90) may be an effective treatment for CLL. We sought to further these data to determine whether the Hsp90 inhibitor, AUY922 (Novartis), is effective against CLL cells in a supportive in vitro environment. AUY922 significantly attenuated changes in immunophenotype and signal transducer and activator of transcription 3 (STAT3) signaling induced by CD40L-fibroblast co-culture but had no effect on the viability of CLL cells in this model. However, AUY922 in combination with fludarabine was significantly more effective at inducing apoptosis in cells in co-culture than either drug alone, an effect that was irrespective of ATM/TP53 dysfunction. In conclusion, our data suggest that further studies and clinical trials of AUY922 in combination with fludarabine may be warranted.

Detection of orphan receptor tyrosine kinase (ROR-1) expression in Egyptian pediatric acute lymphoblastic leukemia.

Receptor tyrosine kinases, a group of tumor-associated antigens, were introduced as targets for cancer intervention strategies. The human orphan receptor tyrosine kinase-1 (ROR-1) is a member of this family. Overexpression of ROR1 has been reported in B-cell chronic lymphocytic leukemia. The aim of this study was to detect the expression profile of ROR1 in 54 pediatric acute lymphoblastic leukemia (ALL) patients. ROR1 was overexpressed in ALL as the ROR1/ ß-actin ratio was higher in ALL children than in control group (P = 0.024). ROR1 is a potential tool for targeted immunotherapy in pediatric ALL patients.

Use of the receptor tyrosine kinase-like orphan receptor 1 (ROR1) as a diagnostic tool in chronic lymphocytic leukemia (CLL).

Flow cytometry is commonly used to establish the diagnosis of chronic lymphocytic leukemia (CLL). A defined combination of antibodies discriminates between normal B cells and CLL cells (coexpression of CD5, CD19, and CD23). The receptor tyrosine-like orphan receptor one (ROR1) is an embryonic glycoprotein involved in several developmental processes. It was shown to be highly and specifically expressed on circulating B lymphoma cells, but not on normal B cells. Here, we examined the potential of ROR1 as a diagnostic marker in initial and follow-up diagnostics of patients with CLL. 105 untreated and 72 treated patients, as well as healthy volunteers were examined using flow cytometry assays. Furthermore, we examined 10 patients with various B cell non-Hodgkin lymphomas (B-NHL). ROR1 was detected using a directly labeled antibody. We detected uniformly high ROR1 expression levels in all CLL samples. In marked contrast, only low or absent ROR1 expression levels were found on B cells from healthy donors. ROR1 expression in CLL patients was not influenced by various treatments. Taken together, ROR1 may be used as a diagnostic marker for CLL. As it is the only antigen which can exclusively be detected on neoplastic B cells it may greatly increase both, specificity as well as sensitivity, in lymphoma diagnostics.