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How basal cell carcinoma (BCC) interacts with its tumor microenvironment to promote growth is unclear. We use singe-cell RNA sequencing to define the human BCC ecosystem and discriminate between normal and malignant epithelial cells. We identify spatial biomarkers of tumors and their surrounding stroma that reinforce the heterogeneity of each tissue type. Combining pseudotime, RNA velocity–PAGA, cellular entropy, and regulon analysis in stromal cells reveals a cancer-specific rewiring of fibroblasts, where STAT1, TGF-β, and inflammatory signals induce a noncanonical WNT5A program that maintains the stromal inflammatory state. Cell-cell communication modeling suggests that tumors respond to the sudden burst of fibroblast-specific inflammatory signaling pathways by producing heat shock proteins, whose expression we validated in situ. Last, dose-dependent treatment with an HSP70 inhibitor suppresses in vitro vismodegib-resistant BCC cell growth, Hedgehog signaling, and in vivo tumor growth in a BCC mouse model, validating HSP70’s essential role in tumor growth and reinforcing the critical nature of tumor microenvironment cross-talk in BCC progression.

How basal cell carcinoma (BCC) interacts with its tumor microenvironment to promote growth is unclear. Here we use singe-cell RNA sequencing to define the human BCC ecosystem and discriminate between normal and malignant epithelial cells. We identify spatial biomarkers of both tumors and their surrounding stroma that reinforce the heterogeneity of each tissue type. Combining pseudotime, RNA velocity, cellular entropy, and regulon analysis in stromal cells reveal a cancer-specific rewiring of fibroblasts where STAT1, TGF-β, and inflammatory signals induce a non-canonical WNT5A program that maintains the stromal inflammatory state. Cell-cell communication modeling suggests that tumors respond to the sudden burst of fibroblast-specific inflammatory signaling pathways by producing heat shock proteins, which we validated in situ. Finally, dose-dependent treatment with an HSP70 inhibitor suppresses in vitro BCC cell growth and Hedgehog signaling and in vivo tumor growth in a BCC mouse model, validating HSP70’s essential role in tumor growth and reinforcing the critical nature of tumor microenvironment crosstalk in BCC progression.

Primary cilia loss is a common feature of advanced cancers. While primary cilia are necessary to initiate Hedgehog (HH)-driven cancers, how HH pathway activity is maintained in advanced cancers devoid of primary cilia is unclear. Here, we find that HH-driven basal cell carcinoma (BCC) accumulate mutations in the Alström and Usher syndrome genes in advanced and SMO inhibitorresistant tumors. Loss of Alström and Usher syndrome gene expression, which are common underlying causes of deafness and blindness, suppresses ciliogenesis and HH signaling. Atypical protein kinase C iota/lambda (aPKC) is a GLI1 kinase with higher expression in advanced BCCs and we show that a constitutively active isoform drives HH pathway activity and mutually antagonizes primary cilia. Overexpression of the constitutively active aPKC variant can maintain HH pathway activity in the absence of primary cilia and can drive resistance to the SMO antagonist vismodegib regardless of cilia status. Finally, superficial BCCs display less primary cilia and higher aPKC expression, which is inversely correlated in nodular BCC subtypes. Our results suggest aPKC may serve as a biomarker for SMO inhibitor sensitivity and a target for clinical application.

Actin polymerization is important to generate primary cilia. Drummond et al. show that upstream actin regulators are necessary for this process by controlling aPKC and Src kinase activity to promote Hedgehog signaling and restrict primary cilia., Primary cilia are polarized organelles that allow detection of extracellular signals such as Hedgehog (Hh). How the cytoskeleton supporting the cilium generates and maintains a structure that finely tunes cellular response remains unclear. Here, we find that regulation of actin polymerization controls primary cilia and Hh signaling. Disrupting actin polymerization, or knockdown of N-WASp/Arp3, increases ciliation frequency, axoneme length, and Hh signaling. Cdc42, a potent actin regulator, recruits both atypical protein pinase C iota/lambda (aPKC) and Missing-in-Metastasis (MIM) to the basal body to maintain actin polymerization and restrict axoneme length. Transcriptome analysis implicates the Src pathway as a major aPKC effector. aPKC promotes whereas MIM antagonizes Src activity to maintain proper levels of primary cilia, actin polymerization, and Hh signaling. Hh pathway activation requires Smoothened-, Gli-, and Gli1-specific activation by aPKC. Surprisingly, longer axonemes can amplify Hh signaling, except when aPKC is disrupted, reinforcing the importance of the Cdc42–aPKC–Gli axis in actin-dependent regulation of primary cilia signaling.

Sporadic and basal cell nevus syndrome basal cell carcinomas show differential response rates to Smoothened inhibitors. Chiang et al. demonstrate notable decreases in UV-induced mutagenesis, total mutation load, genomic instability, and drug-resistant mutations among basal cell nevus syndrome basal cell carcinomas using whole exome sequencing, which may explain the differences in drug response rates.

During tissue development, transcription factors bind regulatory DNA regions called enhancers, often located at great distances from the genes they regulate, to control gene expression. The enhancer landscape during embryonic stem cell differentiation has been well characterized. By contrast, little is known about the shared and unique enhancer regulatory mechanisms in different ectodermally derived epithelial cells. Here we use ChIP sequencing (ChIP-seq) to identify domains enriched for the histone marks histone H3 lysine 4 trimethylation, histone H3 lysine 4 monomethylation, and histone H3 lysine 27 acetylation (H3K4me3, H3K4me1, and H3K27ac) and define, for the first time, the super enhancers and typical enhancers active in primary human corneal epithelial cells. We show that regulatory regions are often shared between cell types of the ectodermal lineage and that corneal epithelial super enhancers are already marked as potential regulatory domains in embryonic stem cells. Kruppel-like factor (KLF) motifs were enriched in corneal epithelial enhancers, consistent with the important roles of KLF4 and KLF5 in promoting corneal epithelial differentiation. We now show that the Kruppel family member KLF7 promotes the corneal progenitor cell state; on many genes, KLF7 antagonized the corneal differentiation-promoting KLF4. Furthermore, we found that two SNPs linked previously to corneal diseases, astigmatism, and Stevens-Johnson syndrome fall within corneal epithelial enhancers and alter their activity by disrupting transcription factor motifs that overlap these SNPs. Taken together, our work defines regulatory enhancers in corneal epithelial cells, highlights global gene-regulatory relationships shared among different epithelial cells, identifies a role for KLF7 as a KLF4 antagonist in corneal epithelial cell differentiation, and explains how two SNPs may contribute to corneal diseases.

During vertebrate somitogenesis, retinoic acid is known to establish the position of the determination wavefront, controlling where new somites are permitted to form along the anteroposterior body axis. Less is understood about how RAR regulates somite patterning, rostral-caudal boundary setting, specialization of myotome subdivisions, or the specific RAR subtype that is required for somite patterning. Characterizing the function of RARβ has been challenging due to the absence of embryonic phenotypes in murine loss-of-function studies. Using the Xenopus system, we show that RARβ2 plays a specific role in somite number and size, restriction of the presomitic mesoderm anterior border, somite chevron morphology and hypaxial myoblast migration. Rarβ2 is the RAR subtype whose expression is most up-regulated in response to ligand and its localization in the trunk somites positions it at the right time and place to respond to embryonic retinoid levels during somitogenesis. RARβ2 positively regulates Tbx3 a marker of hypaxial muscle, and negatively regulates Tbx6 via Ripply2 to restrict the anterior boundaries of the presomitic mesoderm and caudal progenitor pool. These results demonstrate for the first time an early and essential role for RARβ2 in vertebrate somitogenesis.

Retinoic acid receptor gamma 2 (RARγ2) is the major RAR isoform expressed throughout the caudal axial progenitor domain in vertebrates. During a microarray screen to identify RAR targets, we identified a subset of genes that pattern caudal structures or promote axial elongation and are upregulated by increased RARmediated repression. Previous studies have suggested that RAR is present in the caudal domain, but is quiescent until its activation in late stage embryos terminates axial elongation. By contrast, we show here that RARγ2 is engaged in all stages of axial elongation, not solely as a terminator of axial growth. In the absence of RA, RARγ2 represses transcriptional activity in vivo and maintains the pool of caudal progenitor cells and presomitic mesoderm. In the presence of RA, RARγ2 serves as an activator, facilitating somite differentiation. Treatment with an RARγ-selective inverse agonist (NRX205099) or overexpression of dominant-negative RARγ increases the expression of posterior Hox genes and that of marker genes for presomitic mesoderm and the chordoneural hinge. Conversely, when RAR-mediated repression is reduced by overexpressing a dominant-negative co-repressor (c-SMRT), a constitutively active RAR (VP16-RARγ2), or by treatment with an RARγ-selective agonist (NRX204647), expression of caudal genes is diminished and extension of the body axis is prematurely terminated. Hence, gene repression mediated by the unliganded RARγ2-co-repressor complex constitutes a novel mechanism to regulate and facilitate the correct expression levels and spatial restriction of key genes that maintain the caudal progenitor pool during axial elongation in Xenopus embryos. © 2014. Published by The Company of Biologists Ltd.