Receptor Tyrosine Kinase-like Orphan Research Articles

A chemical proteomics approach for global mapping of functional lysines on cell surface of living cell.

Cell surface proteins are responsible for many crucial physiological roles, and they are also the major category of drug targets as the majority of therapeutics target membrane proteins on the surface of cells to alter cellular signaling. Despite its great significance, ligand discovery against membrane proteins has posed a great challenge mainly due to the special property of their natural habitat. Here, we design a new chemical proteomic probe OPA-S-S-alkyne that can efficiently and selectively target the lysines exposed on the cell surface and develop a chemical proteomics strategy for global analysis of surface functionality (GASF) in living cells. In total, we quantified 2639 cell surface lysines in Hela cell and several hundred residues with high reactivity were discovered, which represents the largest dataset of surface functional lysine sites to date. We discovered and validated that hyper-reactive lysine residues K382 on tyrosine kinase-like orphan receptor 2 (ROR2) and K285 on Endoglin (ENG/CD105) are at the protein interaction interface in co-crystal structures of protein complexes, emphasizing the broad potential functional consequences of cell surface lysines and GASF strategy is highly desirable for discovering new active and ligandable sites that can be functionally interrogated for drug discovery.

Theranostic silk sericin/SPION nanoparticles for targeted delivery of ROR1 siRNA: Synthesis, characterization, diagnosis and anticancer effect on triple-negative breast cancer

Triple-negative breast cancer (TNBC) has the worst prognosis among all breast cancer subtypes. The lack of proper treatments prompted scientists to find a practical targeted therapy to treat this type of tumor. Based on previous studies, tyrosine kinase-like orphan receptor (ROR1) is overexpressed in TNBC cells. Here, we designed a system consisting of superparamagnetic iron oxide nanoparticles (SPIONs) decorated with silk sericin (SS NPs) for the targeted delivery of ROR1 siRNA, a gene silencer to knockdown the expression of human ROR1 gene. NPs exhibited spherical shape of about 193 nm with acceptable properties both in vitro and in vivo. The apoptosis study showed significant death of MDA-MB-231 cells after 24 h treatment with the prepared NPs. The real-time PCR study also demonstrated an almost complete shutdown of ROR1 expression. Guided by magnetic field, enhanced accumulation of NPs was observed in breast tumors induced by 4T1 cells in BALB/c mice. Histological evaluation of the tumor exhibited necrosis 14 days post-treatment with the siRNA-loaded NPs; whereas, the untreated tumor was proliferating. Also, the tumor growth rate was significantly decreased after treatment with siRNA-loaded NPs in vivo. In conclusion, the prepared delivery system could be considered as a potential therapeutic strategy for treating TNBC.

Paclitaxel-based supramolecular hydrogel loaded with mifepristone for the inhibition of breast cancer metastasis.

Breast cancer is the leading cause of cancer death among women and almost all of the breast cancer‐caused mortality is related to metastasis. It has been reported that glucocorticoid facilitates the metastasis of breast cancer in mice, and mifepristone can antagonize the effect of glucocorticoid. Paclitaxel is one of the important drugs in the treatment of breast cancer. Mifepristone combined with paclitaxel could be an effective strategy for inhibiting breast cancer metastasis. However, their inherent defects, in terms of short blood circulation half‐life and lack of tumor targeting, not only limit their effectiveness but also cause adverse reactions. Therefore, our aim is to explore a novel protocol against breast cancer metastasis, further optimize its therapeutic efficacy by a nanodelivery system, and explore its mechanism. Herein, a paclitaxel‐conjugated and mifepristone‐loaded hydrogel (PM‐nano) was prepared by self‐assembly. Its characterizations were studied. The antimetastatic effect was evaluated in vitro and in vivo and its mechanism was also explored by western blot assay. The resultant PM‐nano was developed with favorable water solubility and good biocompatibility. Moreover, PM‐nano displayed increased cell uptake properties and stimulated drug release in the tumor micro‐acidic environment. The PM‐nano was more effective in inhibiting the proliferation and metastasis of breast cancer than other groups in vitro and in vivo. The PM‐nano might inhibit metastasis through glucocorticoid receptor/receptor tyrosine kinase‐like orphan receptor 1 and MMPs. Taken together, PM‐nano showed superior antimetastatic effects against breast cancer and excellent biocompatibility in vitro and in vivo, providing a new option for limiting metastasis.

Expression and role of resistin on steroid secretion in the porcine corpus luteum

Resistin plays an important role in adipogenesis, obesity, insulin resistance, and reproduction. Previous studies showed resistin action on ovarian follicular cells; however, whether resistin regulates steroid secretion in luteal cells is still unknown. Our aim was first to determine the expression of resistin and its potential receptors (tyrosine kinase-like orphan receptor 1 (ROR1) and toll-like receptor 4 (TLR4)) in the porcine corpus luteum (CL), regulation of its expression, effect on kinases phosphorylation, and luteal steroidogenesis. Our results showed that the expression of resistin and its receptors was dependent on the luteal phase and this was higher at the mRNA level in the late compared with the early and middle luteal phase. At the opposite, resistin protein expression was higher in the middle and late compared with the early luteal phase, while ROR1 and TLR4 expression was highest in the early luteal phase. Additionally, we observed cytoplasmic localisation of resistin, ROR1, and TLR4 in small and large luteal cells. We found that luteinising hormone, progesterone (P4), insulin, and insulin-like growth factor 1 regulated the protein level of resistin, ROR1, and TLR4. Resistin decreased P4 and increased oestradiol (E2) secretion via changes in steroidogenic enzymes expression and via the activation of protein kinase A (PKA) and mitogen-activated protein kinase (MAP3/1), increased the expression of receptors LHCGR and ESR2 and decreased the expression of PGR. Moreover, resistin decreased PKA phosphorylation and enhanced MAP3/1 phosphorylation. Taken together, resistin could act directly on steroid synthesis and serve as an important factor in in vivo luteal cell function.

Wnt5a/Ror2 pathway contributes to the regulation of cholesterol homeostasis and inflammatory response in atherosclerosis

Atherosclerosis (AS) is characterized by lipids metabolism disorder and inflammatory response. Accumulating evidence has demonstrated that Wingless type 5a (Wnt5a) is implicated in cardiovascular diseases through non-canonical Wnt cascades. However, its precise role during the pathogenesis of AS is still unclear. Therefore, the present study aims to investigate the role and the underlying mechanism of Wnt5a/receptor tyrosine kinase-like orphan receptor 2 (Ror2) pathways in the promotion of AS process through affecting lipid accumulation and inflammation. In atherosclerotic clinical samples, Wnt5a levels were measured by using enzyme-linked immunosorbent assay (ELISA) assay. In vivo experiments were conducted by using apolipoprotein E knockout (apoE−/−) mice model. Vascular smooth muscle cells (VSMCs) were applied for in vitro studies. Wnt5a was highly expressed in both of atherosclerotic clinical samples and apoE−/− mice. The knockdown of Wnt5a significantly inhibited cholesterol accumulation and inflammatory response. Additionally, the lipopolysaccharide (LPS)-induced inflammation aggravated the cholesterol accumulation and decreased adenosine triphosphate (ATP)-binding cassette transporter A1 (ABCA1) expression in VSMCs. Depletion of intracellular cholesterol by β-cyclodextrin (β-CD) led to the upregulation of ABCA1 and the inhibition of inflammation. Conversely, the overexpression of Wnt5a inhibited ABCA1 expression, facilitated cholesterol accumulation, impared cholesterol efflux, promoted NF-κB nuclear translocation and the inflammatory cytokines secretion. Moreover, the knockdown of Ror2 increased ABCA1 expression and reduced Wnt5a-induced cholesterol accumulation and inflammatory responses. Furthermore, the knockdown of ABCA1 enhanced cholesterol accumulation and inflammatory response. Therefore, Wnt5a/Ror2 pathway was critical in regulating cholesterol homeostasis and inflammatory response, which might be a promising therapeutic target for AS therapy.

Higher expression level of tyrosine kinase‑like orphan receptor 2 and Wnt member 5a in papillary thyroid carcinoma is associated with poor prognosis

The tyrosine kinase-like orphan receptor 2 (ROR2) has a wnt-mediated, pro-tumorigenic role in certain types of cancer. The present study was designed to assess the protein expression level of ROR2 and its putative ligand Wnt member 5a (Wnt5a), as well as the association with clinicopathological features in papillary thyroid carcinoma (PTC). A total of 58 patients were recruited, resulting in 58 human PTC tissue samples and their paired adjacent noncancerous tissue samples being obtained. The protein expression levels of ROR2 and Wnt5a were evaluated by immunohistochemistry and western blotting, and messenger RNA expression levels were determined by reverse transcription-quantitative polymerase chain reaction. ROR2 and Wnt5a protein and mRNA expression were significantly overexpressed in PTC tissues (P<0.05). The present study also revealed that ROR2 and Wnt5a were significantly associated with tumor stage and lymph node metastasis (P<0.05). There was a positive association between ROR2 and Wnt5a expression levels (r=0.857; P=0.007). In conclusion, ROR2 and Wnt5a may act as tumor suppressor genes in the development of PTC; overexpression of ROR2 and Wnt5a in PTC may be important for tumorigenesis and tumor development.

IGF1R- and ROR1-Specific CAR T Cells as a Potential Therapy for High Risk Sarcomas.

Patients with metastatic or recurrent and refractory sarcomas have a dismal prognosis. Therefore, new targeted therapies are urgently needed. This study was designed to evaluate chimeric antigen receptor (CAR) T cells targeting the type I insulin-like growth factor receptor (IGF1R) or tyrosine kinase-like orphan receptor 1 (ROR1) molecules for their therapeutic potential against sarcomas. Here, we report that IGF1R (15/15) and ROR1 (11/15) were highly expressed in sarcoma cell lines including Ewing sarcoma, osteosarcoma, alveolar or embryonal rhabdomyosarcoma, and fibrosarcoma. IGF1R and ROR1 CAR T cells derived from eight healthy donors using the Sleeping Beauty (SB) transposon system were cytotoxic against sarcoma cells and produced high levels of IFN-γ, TNF-α and IL-13 in an antigen-specific manner. IGF1R and ROR1 CAR T cells generated from three sarcoma patients released significant amounts of IFN-γ in response to sarcoma stimulation. The adoptive transfer of IGF1R and ROR1 CAR T cells derived from a sarcoma patient significantly reduced tumor growth in pre-established, systemically disseminated and localized osteosarcoma xenograft models in NSG mice. Infusion of IGF1R and ROR1 CAR T cells also prolonged animal survival in a localized sarcoma model using NOD/scid mice. Our data indicate that both IGF1R and ROR1 can be effectively targeted by SB modified CAR T cells and that such CAR T cells may be useful in the treatment of high risk sarcoma patients.