Unbalancing cAMP and Ras/MAPK pathways as a therapeutic strategy for cutaneous neurofibromas
Helena Mazuelas, Miriam Magallón-Lorenz, Itziar Uriarte-Arrazola, Alejandro Negro, Inma Rosas, Ignacio Blanco, Elisabeth Castellanos, Conxi Lázaro, Bernat Gel, Meritxell Carrió, Eduard Serra
Helena Mazuelas, Miriam Magallón-Lorenz, Itziar Uriarte-Arrazola, Alejandro Negro, Inma Rosas, Ignacio Blanco, Elisabeth Castellanos, Conxi Lázaro, Bernat Gel, Meritxell Carrió, Eduard Serra
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Research Article Cell biology Genetics

Unbalancing cAMP and Ras/MAPK pathways as a therapeutic strategy for cutaneous neurofibromas

Abstract

Cutaneous neurofibromas (cNFs) are benign Schwann cell (SC) tumors arising from subepidermal glia. Individuals with neurofibromatosis type 1 (NF1) may develop thousands of cNFs, which greatly affect their quality of life. cNF growth is driven by the proliferation of NF1–/– SCs and their interaction with the NF1+/– microenvironment. We analyzed the crosstalk between human cNF-derived SCs and fibroblasts (FBs), identifying an expression signature specific to the SC-FB interaction. We validated the secretion of proteins involved in immune cell migration, suggesting a role of SC-FB crosstalk in immune cell recruitment. The signature also captured components of developmental signaling pathways, including the cAMP elevator G protein–coupled receptor 68 (GPR68). Activation of Gpr68 by ogerin in combination with the MEK inhibitor (MEKi) selumetinib reduced viability and induced differentiation and death of human cNF-derived primary SCs, a result corroborated using an induced pluripotent stem cell–derived 3D neurofibromasphere model. Similar results were obtained using other Gpr68 activators or cAMP analogs/adenylyl cyclase activators in combination with selumetinib. Interestingly, whereas primary SC cultures restarted their proliferation after treatment with selumetinib alone was stopped, the combination of ogerin-selumetinib elicited a permanent halt on SC expansion that persisted after drug removal. These results indicate that unbalancing the Ras and cAMP pathways by combining MEKi and cAMP elevators could be used as a potential treatment for cNFs.

Authors

Helena Mazuelas, Miriam Magallón-Lorenz, Itziar Uriarte-Arrazola, Alejandro Negro, Inma Rosas, Ignacio Blanco, Elisabeth Castellanos, Conxi Lázaro, Bernat Gel, Meritxell Carrió, Eduard Serra

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Figure 7

Selumetinib-ogerin and selumetinib-PAM71 cotreatments increase cell death in neurofibromaspheres.

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Selumetinib-ogerin and selumetinib-PAM71 cotreatments increase cell deat...
(A) Representative phase-contrast images showing neurofibromasphere morphology differences at –1 hour (nontreated) and at 72 hours after treatment with DMSO-treated (vehicle) spheroids, single treatment, and cotreatments. Neurofibromapheres were generated from NF1–/– iPSC-derived differentiating SCs mixed with human cNF-derived FBs. Scale bar: 100μm. (B) Representative images of Acridine Orange– (ThermoFisher) (live, green) and propidium iodide–stained (dead, red) neurofibromaspheres after 72 hours of drug treatment. Scale bars: 100 μm. (C) Disaggregation index (DI) of neurofibromaspheres in A, showing a higher effect in cotreatments than in single treatments. DI is calculated by measuring disaggregated spheroid area normalized by spheroid area at 72 hours of drug treatment. Data are shown as the median of n ≥ 5 spheroids. One-tailed unpaired t test (**P ≤0.01 and ****P ≤ 0.0001, comparing 4 μM selumetinib with 4 μM cotreatment or 40 μM selumetinib with 40 μM cotreatment). (D) Raw integrity density of death channel of neurofibromaspheres in B, showing a higher effect in cotreatments than in single treatments. Data are shown as the median of n ≥ 3 spheroids. One-tailed unpaired t test (*P ≤ 0.05, comparing 4 μM selumetinib with 4 μM cotreatment or 40 μM selumetinib with 40 μM cotreatment. (E) Representative images of Acridine Orange (live, green), propidium iodide (dead, red) and merge (live and death) at 72 hours after selumetinib and ogerin or PAM71 treatments and cotreatments. (F) Ratio of raw integrity density (RawIntDen) in live and death channels of neurofibromaspheres in E. Data are shown as the median of n ≥ 5 spheroids. One-tailed unpaired t test performing a Holm-Bonferroni post hoc adjustment (*P ≤ 0.05, **P ≤0.01, ***P ≤ 0.001, and ****P ≤ 0.0001). (G) Effect of selumetinib, ogerin, and PAM71 treatment and cotreatment on cell viability in neurofibromaspheres. Cell viability was measured at 72 hours after treatment using Cell Titer Glo 3D viability assay. Two independent experiments were performed and at least 5 spheroids per condition/experiment were used. One-tailed unpaired t test (****P ≤ 0.0001) comparing 40 μM selumetinib with 40 μM selumetinib-ogerin cotreatment or 40 μM selumetinib with 40 μM selumetinib-PAM71 cotreatment. FB, fibroblast; iPSC, induced pluripotent stem cell; NC, neural crest; SC, Schwann cell.