Extracellular vesicle-associated tyrosine kinase-like orphan receptors ROR1 and ROR2 promote breast cancer progression.

BACKGROUND: Extracellular vesicles (EVs) harbor a plethora of different biomolecules, which they can transport across cells. In cancer, tumor-derived EVs thereby support the creation of a favorable tumor microenvironment. So far, EV uptake and cargo delivery into target cells have been regarded as the main mechanisms for the pro-tumoral function of EVs. To test this hypothesis, we investigated the fate of the oncogenic transmembrane Wnt tyrosine kinase-like orphan receptor 1 and 2 (ROR1, ROR2) delivered via distinct EV subpopulations to breast cancer cells and aimed to unravel their impact on tumor progression. METHODS: EVs were isolated by differential ultracentrifugation from cell culture supernatant as well as plasma samples from healthy individuals (n = 27) and breast cancer patients (n = 41). EVs were thoroughly characterized by electron microscopy, nanoparticle tracking analysis, immunoblot, and flow cytometry. ROR transfer to target cells was observed using microscopy-based assays and biodistribution experiments were conducted in syngeneic mice. EV impact on cancer cell migration and invasion was tested in functional assays. RESULTS: We observed that the supernatant of ROR-overexpressing cells was sufficient for transferring the receptors to ROR-negative cells. Analyzing the secretome of the ROR-overexpressing cells, we detected a high enrichment of ROR1/2 on large and small EVs, but not on large oncosomes. Interestingly, the majority of ROR-positive EVs remained attached to the target cell surface after 24 h of stimulation and was quickly removed by treatment with trypsin. Nonetheless, ROR-positive EVs increased migration and invasion of breast cancer cells, even after chemically inhibiting EV uptake, in dependence of RhoA downstream signaling. In vivo, ROR-depleted EVs tended to distribute less into organs prone for the formation of breast cancer metastases. ROR-positive EVs were also significantly elevated in the plasma of breast cancer patients and allowed to separate them from healthy controls. CONCLUSIONS: The oncogenic Wnt receptors ROR1/2 are transferred via EVs to the surface of ROR-negative cancer cells, in which they induce an aggressive phenotype supporting tumor progression. Video Abstract.

WNT11/ROR2 signaling is associated with tumor invasion and poor survival in breast cancer.

BACKGROUND: Breast cancer has been associated with activation of the WNT signaling pathway, although no driver mutations in WNT genes have been found yet. Instead, a high expression of the alternative WNT receptor ROR2 was observed, in particular in breast cancer brain metastases. However, its respective ligand and downstream signaling in this context remained unknown. METHODS: We modulated the expression of ROR2 in human breast cancer cells and characterized their gene and protein expression by RNA-Seq, qRT-PCR, immunoblots and reverse phase protein array (RPPA) combined with network analyses to understand the molecular basis of ROR2 signaling in breast cancer. Using co-immunoprecipitations, we verified the interaction of ROR2 with the identified ligand, WNT11. The functional consequences of WNT11/ROR2 signaling for tumor cell aggressiveness were assessed by microscopy, impedance sensing as well as viability and invasion assays. To evaluate the translational significance of our findings, we performed gene set enrichment, expression and survival analyses on human breast cancer brain metastases. RESULTS: We found ROR2 to be highly expressed in aggressive breast tumors and associated with worse metastasis-free survival. ROR2 overexpression induced a BRCAness-like phenotype in a cell-context specific manner and rendered cells resistant to PARP inhibition. High levels of ROR2 were furthermore associated with defects in cell morphology and cell-cell-contacts leading to increased tumor invasiveness. On a molecular level, ROR2 overexpression upregulated several non-canonical WNT ligands, in particular WNT11. Co-immunoprecipitation confirmed that WNT11 indeed interacts with the cysteine-rich domain of ROR2 and triggers its invasion-promoting signaling via RHO/ROCK. Knockdown of WNT11 reversed the pro-invasive phenotype and the cellular changes in ROR2-overexpressing cells. CONCLUSIONS: Taken together, our study revealed a novel auto-stimulatory loop in which ROR2 triggers the expression of its own ligand, WNT11, resulting in enhanced tumor invasion associated with breast cancer metastasis.

The WNT/ROR Pathway in Cancer: From Signaling to Therapeutic Intervention.

The WNT pathway is one of the major signaling cascades frequently deregulated in human cancer. While research had initially focused on signal transduction centered on ß-catenin as a key effector activating a pro-tumorigenic transcriptional response, nowadays it is known that WNT ligands can also induce a multitude of ß-catenin-independent cellular pathways. Traditionally, these comprise WNT/planar cell polarity (PCP) and WNT/Ca2+ signaling. In addition, signaling via the receptor tyrosine kinase-like orphan receptors (RORs) has gained increasing attention in cancer research due to their overexpression in a multitude of tumor entities. Active WNT/ROR signaling has been linked to processes driving tumor development and progression, such as cell proliferation, survival, invasion, or therapy resistance. In adult tissue, the RORs are largely absent, which has spiked the interest in them for targeted cancer therapy. Promising results in preclinical and initial clinical studies are beginning to unravel the great potential of such treatment approaches. In this review, we summarize seminal findings on the structure and expression of the RORs in cancer, their downstream signaling, and its output in regard to tumor cell function. Furthermore, we present the current clinical anti-ROR treatment strategies and discuss the state-of-the-art, as well as the challenges of the different approaches.