Recovery of taste organs and sensory function after PNAS PLUS severe loss from Hedgehog/Smoothened inhibition with cancer drug sonidegib Archana Kumaria, Alexandre N. Ermilovb, Marina Grachtchoukb, Andrzej A. Dlugoszb,c,1, Benjamin L. Allenc,1, Robert M. Bradleya,1, and Charlotte M. Mistrettaa,1,2 aDepartment of Biologic and Materials Sciences, University of Michigan School of Dentistry, Ann Arbor, MI 48109; bDepartment of Dermatology, University of Michigan Medical School, Ann Arbor, MI 48109; and cDepartment of Cell and Developmental Biology, University of Michigan Medical School, Ann Arbor, MI 48109 Edited by Linda M. Bartoshuk, University of Florida, Gainesville, FL, and approved October 11, 2017 (received for review July 19, 2017) Striking taste disturbances are reported in cancer patients treated signaling in FP and circumvallate (CV) papillae and tested the with Hedgehog (HH)-pathway inhibitor drugs, including sonidegib hypothesis that morphologic and functional recovery would occur (LDE225), which block the HH pathway effector Smoothened (SMO). following cessation of HH/SMO inhibition by sonidegib treatment. We tested the potential for molecular, cellular, and functional Taste cells turn over continuously in adults (9, 10), but TB are recovery in mice from the severe disruption of taste-organ biology eliminated when essential signaling pathways, including HH, are and taste sensation that follows HH/SMO signaling inhibition. disrupted (11–13). However, the taste cells are resilient in specific Sonidegib treatment led to rapid loss of taste buds (TB) in both contexts, as illustrated by degeneration after nerve cut in adult fungiform and circumvallate papillae, including disruption of TB mammals and regeneration as nerves grow back into the taste progenitor-cell proliferation and differentiation. Effects were selec- papilla epithelium (14). The taste organ therefore is ideally suited tive, sparing nontaste papillae. To confirm that taste-organ effects of to explore fundamental questions about the biology of tissue ho- sonidegib treatment result from HH/SMO signaling inhibition, we meostasis and renewal, the mechanisms that sustain sensation in studied mice with conditional global or epithelium-specific Smo dele- constantly renewing organs comprised of heterogeneous cell types, CELL BIOLOGY tions and observed similar effects. During sonidegib treatment, and the potential restoration of organs and function after TB chorda tympani nerve responses to lingual chemical stimulation were elimination resulting from deregulation of essential controls. maintained at 10 d but were eliminated after 16 d, associated with Although the HH pathway is a major regulator in several organ nearly complete TB loss. Notably, responses to tactile or cold stimulus systems (15), there is sparse fundamental knowledge about the modalities were retained. Further, innervation, which was maintained function and timing of HH signaling in taste organs, especially the in the papilla core throughout treatment, was not sufficient to sustain mechanisms of cell regulation, the regulation across anterior and TB during HH/SMO inhibition. Importantly, treatment cessation led to posterior tongue tissues, and the effects on taste sensation per se. Whereas transcriptional-level regulation of HH/GLI signaling has rapid and complete restoration of taste responses within 14 d asso- Gli2 ciated with morphologic recovery in about 55% of TB. However, been studied by genetic manipulation of (11), the conse- although taste nerve responses were sustained, TB were not restored quences of HH signal disruption at the cell surface remain largely in all fungiform papillae even with prolonged recovery for several unexplored, although most pharmacologic HH inhibitors act at this level (16). months. This study establishes a physiologic, selective requirement for HH/SMO signaling in taste homeostasis that includes potential for sensory restoration and can explain the temporal recovery Significance after taste dysgeusia in patients treated with HH/SMO inhibitors. Hedgehog pathway-inhibitor drugs effectively treat basal cell taste receptor cell | fungiform papilla | circumvallate papilla | carcinoma, a common skin cancer. However, many patients taste regeneration | chorda tympani nerve taking such drugs report severe taste disturbances that impair their quality of life. To understand the biology behind these ancer patients treated with Hedgehog (HH) pathway in- adverse effects, we studied the consequences of Hedgehog Chibition (HPI) drugs experience severe taste disturbances (1– pathway inhibition on taste organs and neural sensation in mice. 5). The Food and Drug Administration-approved HPI drug Taste bud progenitor-cell proliferation and differentiation were sonidegib (LDE225) blocks HH signaling at the Smoothened altered, resulting in taste bud loss. Nerve responses to lingual taste stimuli were also eliminated, while responses to touch and (SMO) receptor (Fig. 1A) (6). We had reported that oral gavage cold stimuli remained. After stopping Hedgehog pathway in- with sonidegib in mice for 16 or 28 d results in progressive fungi- hibition, taste buds and sensory responses recovered. This study form papilla (FP) taste-organ disruption, taste bud (TB) loss, and advances our understanding of Hedgehog signaling in taste elimination of taste nerve responses to chemical stimuli (7). Now homeostasis and the reported taste recovery after clinical we present data for the initial time course of HPI effects on taste treatments with Hedgehog pathway-inhibiting drugs. organsandcelltypesandonsensation;studyofSmo deletion; and the presence of the HH ligand in the nerve fibers of taste organs. Author contributions: A.K., A.N.E., A.A.D., B.L.A., R.M.B., and C.M.M. designed research; Importantly, the potential for and nature of recovery from HPI A.K., A.N.E., R.M.B., and C.M.M. performed research; M.G. contributed new reagents/ effects in taste organs and taste neurophysiology are demonstrated. analytic tools; A.K., A.N.E., A.A.D., B.L.A., R.M.B., and C.M.M. analyzed data; and A.K., Lingual taste organs are composed of (i) the TB, about 80– A.A.D., B.L.A., R.M.B., and C.M.M. wrote the paper. 100 modified epithelial/neuroepithelial cells of several types; The authors declare no conflict of interest. (ii) the sensory innervation to the TB and surrounding epithe- This article is a PNAS Direct Submission. lium; and (iii) the papilla residence, a stratified epithelium over a Published under the PNAS license. core of stromal cells and connective tissue elements (8). Precise 1A.A.D., B.L.A., R.M.B., and C.M.M. contributed equally to this work. controls within each compartment are essential to integrate the 2To whom correspondence should be addressed. Email: [email protected]. cell processes that support taste organs and sensation. Here we This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. investigated homeostatic lingual taste organ regulation by HH/SMO 1073/pnas.1712881114/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1712881114 PNAS Early Edition | 1of10 Downloaded by guest on September 25, 2021 Fig. 1. Sonidegib alters FP and TB morphology and reduces all TB cell types. (A)TheHHpathwayinOFF and ON signaling and INHIBITED at SMO by sonidegib. (B) Schematic: FP and filiform papillae with HH sig- naling components. (C) H&E staining for FP/TB cate- gories. Dotted lines demarcate TB or regions of TB remnants. Blue dashed surface lines indicate epithelial keratinization in FP; a red dashed line indicates epi- thelial keratinization in filiform papilla in type III atypical FP/no TB. (Scale bar: 50 μm.) (D)Percentageof FP/TB categories: typical FP/TB (category I), atypical FP/ TB (category II), and atypical FP/no TB (category III), after vehicle or sonidegib treatment. Bars are mean ± SEM. Numbers of tongues per group are in paren- theses. Brackets indicate significant differences for treatment durations (two-way ANOVA and Tukey’s HSD post hoc tests); ###P < 0.001 for vehicle vs. soni- degib treatments. Complete F and P values are given in Fig. S1A.(E) Antibody detection of TB cell types: NTPdase2 (type 1), PLCβ2 (type 2), SNAP25 (type 3) after vehicle or sonidegib treatment. Cell nuclei are identified with DAPI (blue). (Insets) TB cell types 2 and 3 were eliminated after 28 d of sonidegib treatment. Type 1 cells (NTPdase2) were eliminated after 36 d. (Scale bar: 50 μm.) (F) Number of TB cell types after 16 or 28 d of sonidegib treatment compared with vehicle treatment. Data are mean ± SEM. Numbers of tongues are in parentheses in bars. Brackets indicate significant differences (one-way ANOVA with Tukey’s HSD post hoc tests). Complete F and P values are given in Fig. S1C. SMO is the core signal transduction component of HH signaling tion of oral sensory modalities of taste, touch, and temperature, (Fig. 1A) (17, 18). In HH signaling, pathway activity is repressed in and the recovery of taste organs and sensation. Our data provide the absence of ligand via the transmembrane receptor PTCH, insight into the regenerative biology and clinical consequences in which represses the pivotal HH signaling protein SMO (19–21). patients treated with sonidegib who experience dysgeusia. HH binding to PTCH1 blocks its inhibition of SMO; downstream signaling is then initiated, resulting in the modulation of GLI Results transcription factors (GLI1, GLI2, GLI3) and leading to the Treatment with HPI Drug Sonidegib Alters FP Taste-Organ Morphology transcription of target genes, including Gli1 and Ptch1 (Fig. 1A). Within 10 D. Before testing recovery from HPI drug treatment, In the tongue, HH pathway components are in distinct compart- it was important first to determine the temporal aspects of ments of adult taste papillae and TB (Fig. 1B), including actively HH/SMO signaling inhibition in mice gavaged with sonidegib for proliferating TB progenitor cells (8, 13). The secreted SHH ligand 5–36 d. We quantified effects by characterizing FP and TB mor- in epithelium is produced principally within TB cells (13, 22), phology as category I (typical FP/TB), II (atypical FP/TB), or III whereas the HH targets, Ptch1 and Gli1, are expressed within (atypical FP/no TB) (Fig.