Neurofibromatosis type 1 (NF1) is characterized by nerve tumors called neurofibromas, in which Schwann cells (SCs) show deregulated RAS signaling. NF1 is also implicated in regulation of cAMP. We identified the G-protein-coupled receptor (GPCR) P2RY14 in human neurofibromas, neurofibroma-derived SC precursors (SCPs), mature SCs and mouse SCPs. Mouse Nf1-/-SCP self-renewal was reduced by genetic or pharmacological inhibition of P2RY14. In a mouse model of NF1, genetic deletion of P2RY14 rescued low cAMP signaling, increased mouse survival, delayed neurofibroma initiation, and improved SC Remak bundles. P2RY14 signals via Gi to increase intracellular cAMP, implicating P2RY14 as a key upstream regulator of cAMP. We found that elevation of cAMP by either blocking the degradation of cAMP or by using a P2RY14 inhibitor diminished NF1-/-SCP self-renewal in vitro and neurofibroma SC proliferation in in vivo. These studies identifyP2RY14 as a critical regulator of SCP self-renewal, SC proliferation and neurofibroma initiation.
The data sets and original figures generated during this study will be available at Synapse Project (https://www.synapse.org/).
Understanding the role of purinergic signaling on tumor formation in a mouse model of Nf1Synapse, doi.org/10.7303/syn27752640.
- Jennifer Patritti Cram
- Jennifer Patritti Cram
- Nancy Ratner
- Nancy Ratner
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Animal experimentation: This study was performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All of the animals were handled according to approved institutional animal care and use committee (IACUC) protocols (#2018-0103 expiration 01-2022) of Cincinnati Children's Hospital. The protocol was approved by the Committee on the Ethics of Animal Experiments of the Cincinnati Children's Hospital.
- William C Hahn, Dana-Farber Cancer Institute, United States
© 2022, Patritti Cram et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
Drug resistance is a challenge in anticancer therapy. In many cases, cancers can be resistant to the drug prior to exposure, i.e., possess intrinsic drug resistance. However, we lack target-independent methods to anticipate resistance in cancer cell lines or characterize intrinsic drug resistance without a priori knowledge of its cause. We hypothesized that cell morphology could provide an unbiased readout of drug resistance. To test this hypothesis, we used HCT116 cells, a mismatch repair-deficient cancer cell line, to isolate clones that were resistant or sensitive to bortezomib, a well-characterized proteasome inhibitor and anticancer drug to which many cancer cells possess intrinsic resistance. We then expanded these clones and measured high-dimensional single-cell morphology profiles using Cell Painting, a high-content microscopy assay. Our imaging- and computation-based profiling pipeline identified morphological features that differed between resistant and sensitive cells. We used these features to generate a morphological signature of bortezomib resistance. We then employed this morphological signature to analyze a set of HCT116 clones (five resistant and five sensitive) that had not been included in the signature training dataset, and correctly predicted sensitivity to bortezomib in seven cases, in the absence of drug treatment. This signature predicted bortezomib resistance better than resistance to other drugs targeting the ubiquitin-proteasome system. Our results establish a proof-of-concept framework for the unbiased analysis of drug resistance using high-content microscopy of cancer cells, in the absence of drug treatment.
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