AP-1 and TGFß cooperativity drives non-canonical Hedgehog signaling in resistant basal cell carcinoma

Abstract

Tumor heterogeneity and lack of knowledge about resistant cell states remain a barrier to targeted cancer therapies. Basal cell carcinomas (BCCs) depend on Hedgehog (Hh)/Gli signaling, but can develop mechanisms of Smoothened (SMO) inhibitor resistance. We previously identified a nuclear myocardin-related transcription factor (nMRTF) resistance pathway that amplifies noncanonical Gli1 activity, but characteristics and drivers of the nMRTF cell state remain unknown. Here, we use single cell RNA-sequencing of patient tumors to identify three prognostic surface markers (LYPD3, TACSTD2, and LY6D) which correlate with nMRTF and resistance to SMO inhibitors. The nMRTF cell state resembles transit-amplifying cells of the hair follicle matrix, with AP-1 and TGFß cooperativity driving nMRTF activation. JNK/AP-1 signaling commissions chromatin accessibility and Smad3 DNA binding leading to a transcriptional program of RhoGEFs that facilitate nMRTF activity. Importantly, small molecule AP-1 inhibitors selectively target LYPD3+/TACSTD2+/LY6D+ nMRTF human BCCs ex vivo, opening an avenue for improving combinatorial therapies.

Fig. 1. nMRTF BCC chromatin accessibility resembles hair follicle matrix.

a IF representative images of MRTF cellular localization in healthy mouse skin, in comparison to localization of HHIP (Hh-responsive matrix transit-amplifying cells), Keratin-14 (interfollicular epidermis and outer root sheath), and phospho-Smad2/3 (TGFß-responsive dermal papilla and matrix transit-amplifying cells). Inset areas (IFE top row, HF matrix bottom row) denoted by white dotted boxes. Single-color images are all derived from the same slide except for pSmad2/3, due to limitations of antibody co-staining. Images are representative of n > 50 hair follicles examined. Scale bar at low power = 50 μm, inset = 25 μm. b IF images of MRTF localization in clinical biopsies of basal cell carcinoma with nuclear or cytoplasmic MRTF (BCC-nMRTF or -cMRTF), pilomatricomas (PMX), epithelial inclusion cysts (EIC), and pilar cysts (PC). Images are representative of n = 3 patient tumors per subtype. Scale bar = 50 μm. c Principal component analysis (PCA) plot showing the relationship of ATAC-seq profiles between resistant BCC cell line ASZ001 (res-BCC), sensitive murine BCC (sens-BCC), basal transit-amplifying cells of hair follicle (basal TAC), bulge hair follicle stem cells (Bulge), hair germ (HG), interfollicular epidermal stem cells (IFE), and suprabasal transit-amplifying cells of the hair follicle (suprabasal TAC). See Supplementary Table 1 for data sources. d IF images of MRTF, Ki67, and Krt14 in mouse hair follicle skin explants treated with DMSO vehicle or 150 μM MRTFⁱ CCG-1423 for 72 h. Scale bar = 20 μm. e Quantification of Ki67+ cells per hair follicle in (d). n = 4 pairs of explants from three mice. ***p < 0.0001. f Representative H&E staining of DMSO or CCG-1423 treated mouse skin explants. Scale bar = 100 μm. g Quantification of hair follicle lengths in (f). Each point represents the length of one HF averaged over up to three separate measurements. **p = 0.0017. All error bars represent mean +/− SD. p-values were calculated using unpaired, two-tailed Student’s t-test.

Fig. 2. LYPD3/TACSTD2/LY6D mark the nMRTF subpopulation in patient BCCs.

a tSNE plot of unbiased clustering of tumor epithelia from 4 naive human BCC tumor scRNA-Seq datasets post multi-CCA alignment, filtered for positive Krt14 expression. b MRTF signature enrichment score (generated from genes in Supplementary Data 1) overlaid on tSNE clusters from (a). c Clusters ranked by mean MRTF signature enrichment score, then grouped as follows: clusters 1–3 = high MRTF, clusters 4–6 = med high, clusters 7–9 = med low, clusters 10–12 = low MRTF. d Heatmap of top 50 genes most enriched in high MRTF group (clusters 1–3) vs. low MRTF group (clusters 10–12). e Violin plots of gene expression levels per group for surface markers LYPD3, TACSTD2, and LY6D. f Representative IF images of surface marker expression in human and mouse MRTF-nuclear vs. MRTF-cytoplasmic BCC regions or HF matrix of normal mouse skin. scale bar = 50 μm. g Quantification of LYPD3, TACSTD2, and LY6D fluorescence intensity normalized to DAPI. Each point represents mean pixel intensity, normalized to mean DAPI intensity, averaged over at least three 100 × 100 μm microscopy fields. **p < 0.01, ***p < 0.001. h Quantification of LYPD3, TACSTD2, and LY6D fluorescence intensity normalized to DAPI of naive patient BCC explants treated with 10 μM MRTFⁱ CCG-1423 for 24 h. Each point represents mean pixel intensity, normalized to mean DAPI intensity, averaged over at least three 100 × 100 μm microscopy fields. *p = 0.0384, **p = 0.0072, ***p = 0.0002. All error bars represent mean  +/− SD. p-values calculated using unpaired, two-tailed Student’s t-test. i Relative reduction in Gli1 mRNA levels as measured by qRT-PCR vs. quantified fluorescence intensity of surface markers normalized to DAPI in naive human BCC (huBCC) explants treated with 1 μM vismodegib for 24 h. Respective linear regression r² values shown in matching colors. j Representative FACS plots showing distributions of ItgA6 (CD49f), LYPD3, TACSTD2 (TROP2), and LYPD3 protein expression in naive human BCC tumors. Final sorted populations taken for further analysis outlined in red and blue. k Gene Set Enrichment Analysis (GSEA) plot of MRTF signature gene list comparing RNA-seq of sorted surface marker positive (SM+) vs. negative (SM−) BCC cells. n = 4 replicates from two tumors. ES = enrichment score, p = nominal p-value. l Gene expression heatmap from GSEA in (k).

Fig. 3. Coincident AP-1 and TGFß signaling are required for BCC resistance.

a Top Gene Onotology (GO) Biological Process terms enriched in upregulated genes of SM+ vs. SM− huBCC cells by Binding and Expression Target Analysis (BETA) integration of RNA-seq and ATAC-seq. p-values calculated using Fisher exact test. b Transcription factors with highest combined ChIP-X Enrichment Analysis (ChEA) score enriched in SM+ vs. SM− huBCC cells by BETA integration of RNA-seq and ATAC-seq. c Transcription factor motifs enriched in differentially open chromatin peaks in SM+ vs. SM− huBCC cells by BETA integration of RNA-seq and ATAC-seq. d Gli1 qRT-PCR in resistant BCC cell line ASZ001 treated with T5224 (AP-1 inhibitor) or SB431542 (ALK5 inhibitor) for 24 h, normalized to DMSO control. Vertical dotted line represents published IC50 in culture. e Cell viability of resistant BCC cell line ASZ001 treated with inhibitors for 72 h, measured by MTS assay. f Gli1 qRT-PCR of resistant BCC cell line ASZ001 transfected with siRNAs against AP-1 and TGFß family genes for 48 h, compared to universal negative control siRNA. Each pair of matching-colored bars represents two distinct siRNA oligos per target gene. **p < 0.01, ***p < 0.001. g Rho G-LISA assay with negative control cells serum starved for 24 h, and positive control cells stimulated with 10% FBS for 5 min. ***p < 0.001. Open circles on all bar graphs represent independent biological replicates. h IF staining of resistant BCC cell line ASZ001 treated with DMSO, 20 μM T-5224 (AP-1 inhibitor), or 10 µM SB431542 (ALK5 inhibitor). Scale bar = 25 μm. i Quantification of MRTF fluorescence intensity across cell radius in (h), measured as µm distance from cell center. Nuclear boundaries represented as DAPI intensity. Mean intensities of n > 50 cells shown as solid lines, with SEM as dotted lines. j MTS cell viability assay of resistant BCC cell line ASZ001 treated with increasing doses of vismodegib (SMOⁱ) only, or vismodegib + 10 μM SB431542 (ALK5 inhibitor) or 5 μM T5224 (AP-1 inhibitor). ***p < 0.001. All error bars represent mean +/− SD. p-values calculated using unpaired, two-tailed Student’s t-test.

Fig. 4. AP-1 and Smad3 induce transcription of Rho regulators including GEFs.

a Heatmap of differentially expressed genes as measured by RNA-seq of resistant ASZ001 cells treated with 10 μM SB431542 (ALK5 inhibitor) or 20 μM T5224 (AP-1 inhibitor). b Overlap between AP-1 and TGFß dependent genes by RNA-seq, defined as log₂ FC < −1 and p > 0.05 in inhibitor-treated cells. Genes listed in Supplementary Data 2. c GO Molecular function terms enriched in genes dependent on both AP-1 and TGFß signaling. P-values calculated by Fisher exact test. d Gli1 qRT-PCR of resistant BCC cell line ASZ001 transfected with siRNAs targeting selected GEFs for 48 h. Each pair of matching-colored bars represents two distinct siRNA oligos per target gene. *p < 0.05, **p < 0.01. e IF images of MRTF and Arhgef17 protein expression in murine sensitive (cMRTF) and resistant (nMRTF) BCCs. Scale bar = 50 μm. f Quantification of (e) as measured by Arhgef17 fluorescence intensity vs. DAPI. Each point represents an individual tumor, mean pixel intensity normalized to mean DAPI intensity quantified over at least three 100 × 100 μm microscopy fields. *p = 0.0107. g IF images of MRTF protein localization in resistant BCC cell line ASZ001 transfected with Cy-3 conjugated siRNAs. Scale bar = 25 μm. h Quantification of MRTF fluorescence intensity across cell radius in (g), measured as µm distance from cell center. Nuclear boundaries represented as DAPI intensity. Mean intensities of n > 50 cells shown as solid lines, with SEM as dotted lines. i Diagram describing putative signaling pathway and corresponding small-molecule inhibitors. j Epistatic studies measured by Gli1 qRT-PCR of resistant BCC cell line ASZ001 transfected with overexpression constructs and treated with inhibitors. ***p < 0.001. Open circles on all bar graphs represent independent biological replicates. All error bars represent mean +/− SD. P-values calculated using unpaired, two-tailed Student’s t-test.

Fig. 5. JunD/AP-1, but not TGFß, is sufficient to drive nMRTF.

a Enhancement of Hh signaling in NIH-3T3 cells measured by Gli1 qRT-PCR transiently transfected with overexpression constructs for 48 h and treated with 30 μM Smoothened agonist (SAG) for 24 h. **p < 0.01. b qRT-PCR of various target genes in 3T3s transiently transfected with GFP control vectorwith or without 5 ng/ml TGFß ligand supplementation, or JunD overexpression construct. ***p < 0.001. c Gli1 qRT-PCR in 3T3 cells transiently transfected with GFP control vector or JunD and treated with 30 μM Smoothened agonist (SAG) for 24 h. ***p < 0.0001. d RhoA activation quantified by G-LISA assay in 3T3 cells transiently transfected with GFP control or overexpression constructs for 48 h. **p < 0.01. Open circles on all bar graphs represent independent biological replicates. All error bars represent mean +/− SD. P-values calculated using unpaired, two-tailed Student’s t-test. e IF images of 3T3 cells transfected with HA-tagged MRTF construct with or without 5 ng/ml recombinant TGFß ligand supplementation, Arhgef17 or JunD overexpression construct. Scale bar = 25 μm. f Quantification of MRTF intensity across cell radius in (e), measured as µm distance from cell center. Nuclear boundaries represented as DAPI intensity. Mean intensities of n > 50 cells shown as solid lines, with SEM as dotted lines.

Fig. 6. AP-1 establishes the Smad3 DNA binding profile of resistant BCC.

a Heatmap and line graphs of ATAC-seq signal in resistant ASZ001 cells treated with and without 20 μM AP-1 inhibitor T5224 across pSmad3 ChIP binding sites. b Overlap of genes displaying loss of chromatin accessibility (ATAC-seq), Smad3 binding (ChIP-seq), and/or expression (RNA-seq) in response to AP-1 inhibitor treatment in resistant BCC cells, defined as log₂ FC < −1 and p > 0.05. Genes listed in Supplementary Data 3. c GO molecular function terms enriched in genes showing AP-1 dependence of chromatin accessibility, Smad3 binding, and mRNA expression levels. P-values calculated by Fisher exact test. d Visualization of ATAC and ChIP peaks at Arhgef17 regulatory locus. ATAC peak of interest has been highlighted, and scissors represent target sites for CRISPR guide RNAs. e List of RhoGEFs dependent on AP-1 by chromatin accessibility, Smad3 binding, and mRNA expression levels in resistant BCC cells. f Arhgef17 qRT-PCR in WT ASZ cells and Arhgef17 ATAC-peak deletion (Arhgef17^AD) cell line. ***p < 0.0001. g Gli1 qRT-PCR in WT and Arhgef17^AD ASZ cell lines, treated with 5 ng/ml of TGFß3 ligand for 24 h or 1 μg/ml of Rho activator II for 6 h. *p = 0.0344, ***p = 0.0003. h Gli1 qRT-PCR in WT and Arhgef17^AD ASZ cell lines transiently transfected with overexpression constructs. ***p = 0.0002. Open circles on all bar graphs represent independent biological replicates. All error bars represent mean +/− SD. P-values calculated using unpaired, two-tailed Student’s t-test. i Gli1 qRT-PCR in WT and Arhgef17^AD ASZ cell lines, treated with increasing dosages of vismodegib or CCG-1423. Calculated IC50 of ASZ WT shown in blue, ARHGEF17^AD in red. **p < 0.01, ***p < 0.001. j IF images of MRTF protein localization and actin polymerization in WT ASZ and Arhgef17^AD cell lines. k Quantification of MRTF fluorescence intensity across cell radius in (j), measured as µm distance from cell center. Nuclear boundaries represented as DAPI intensity. Mean intensities of n > 50 cells shown as solid lines, with SEM as dotted lines. l Correlation of relative reduction in Gli1 mRNA expression of human tumor explants treated with 40 μM AP-1 inhibitor T-5224 for 24 h, with their relative intensity of surface marker immunostaining. Tumors were categorized as nuclear MRTF if immunofluorescent staining of MRTF colocalizing with DAPI was present in at least one of four separate 200 μm × 200 μm microscopy fields, otherwise they were categorized as cytoplasmic MRTF.