Targeted CRISPR approach reveals an essential role for neuropeptide Y receptor Y5 in Ewing sarcoma extrapulmonary
Mina Adnani1, Sung-Hyeok Hong1, Susana Galli1
1Department of Biochemistry and Molecular & Cellular Biology, Georgetown University, Washington, DC, USA.
Abstract:
Ewing sarcoma (ES) is a pediatric malignancy that lacks adequate therapies for its metastatic form. These tumors constitutively express neuropeptide Y (NPY) and its Y5 receptor (Y5R), which leads to elevated levels of the peptide in patients' serum. In animal models, xenografts secreting NPY metastasize to extrapulmonary niches, including bone; the phenotype associated with adverse prognosis in ES patients. To determine the role of the NPY/Y5R axis in ES extrapulmonary dissemination, we used a doxycycline-inducible CRISPR/Cas9 system to knockout Y5R in SK-ES-1 xenografts that metastasize to these niches. We have shown that metastases developing from heterogenous SK-ES-1/Y5R-sgRNA primary tumors in doxycycline-treated mice were initiated exclusively by SK-ES-1 clones with a functional NPY5R gene. Similarly, metastasis from wild type SK-ES-1 xenografts was associated with a selection of clones with NPY5R gene gain. In vitro assays identified Y5R-dependent ES cell motility driven by RhoA activation as the mechanism underlying the metastatic effects of NPY. In ES cell lines that secrete NPY, the autocrine NPY/Y5R loop was responsible for maintaining basal cell motility, while ES cells that do not release the peptide responded to the exogenous NPY. These data provide evidence for the crucial role of the NPY/Y5R axis in ES metastasis.
Insights
The neuropeptide Y (NPY) and its Y5 receptor (Y5R) axis drives Ewing sarcoma (ES) metastasis. Targeting this axis may offer new therapeutic strategies for metastatic ES, a pediatric cancer with limited treatment options.
Area of Science:
- Oncology
- Molecular Biology
- Cancer Metastasis Research
Background:
- Ewing sarcoma (ES) is a pediatric cancer with poor outcomes for metastatic disease.
- Neuropeptide Y (NPY) and its Y5 receptor (Y5R) are constitutively expressed in ES tumors, leading to elevated serum NPY levels.
- NPY-secreting xenografts in animal models show extrapulmonary metastasis, a poor prognostic indicator in ES patients.
Purpose of the Study:
- To investigate the role of the NPY/Y5R axis in the extrapulmonary dissemination of ES.
- To elucidate the molecular mechanisms by which NPY/Y5R signaling contributes to ES cell motility and metastasis.
Main Methods:
- Utilized a doxycycline-inducible CRISPR/Cas9 system to knockout Y5R in ES xenografts.
- Analyzed metastatic initiation from heterogeneous primary tumors in vivo.
- Performed in vitro assays to assess ES cell motility and RhoA activation.
- Investigated autocrine and paracrine roles of the NPY/Y5R axis in ES cell lines.
Main Results:
- Metastases from heterogeneous tumors were initiated exclusively by ES clones with a functional NPY5R gene.
- Metastasis from wild-type xenografts showed selection for clones with NPY5R gene gain.
- Y5R signaling directly drives ES cell motility via RhoA activation.
- An autocrine NPY/Y5R loop maintains basal motility in NPY-secreting ES cells, while exogenous NPY stimulates motility in non-secreting cells.
Conclusions:
- The NPY/Y5R axis plays a critical role in ES extrapulmonary metastasis.
- Targeting the NPY/Y5R pathway represents a potential therapeutic strategy for metastatic ES.
- Understanding the NPY/Y5R signaling in ES may lead to improved treatment outcomes for pediatric cancer patients.
