Fig. 4: ANKRD1 regulates a CAF transcriptional program of clinical significance.

a Four different primary CAFs were infected with two shRNA targeting ANKRD1 (shANKRD1#1, #2) or shRNA control (shCTRL) were examined by cDNA microarray hybridization and analyzed using the Transcriptomic Analysis Console software (TAC), p-values were calculated by two-way ANOVA test. Volcano-Plot of differentially expressed genes (DEG) was generated by filtering for genes downmodulated or upregulated by shANKRD1 (FC > 1.5, p value < 0.05 and FC < −1.5, p value < 0.05, respectively). Black dotted lines separate genes filtered by FC (x-axis) and p.value (y-axis). b Gene Set Enrichment Analysis (GSEA) of skin CAF signature, myofibroblastic CAF (myCAF) and inflammatory CAF (iCAF) were applied to ANKRD1 silenced and control CAFs. c GSEA analysis of ANKRD1-silenced CAF profile using deposited gene sets of CAF-related pathways. Signatures were downloaded from the GSEA web page (http://www.gsea-msigdb.org/gsea/msigdb/human/genesets.jsp), and the TGFB signature was downloaded from GSE79621. d Cell type expression analysis using the ENRICHR tool (https://maayanlab.cloud/Enrichr/). Analysis was performed by computing genes downmodulated by shANKRD1 (FC < −2, <0.05 p value, measured with two-way ANOVA) into an extensive collection of RNA-seq data sets available through the ARCHS4 web resource (https://maayanlab.cloud/archs4/). The bar graph shows the cell type for which the genes are enriched. Values are expressed as –Log10 (p value). All the genes regulated by ANKRD1 and expressed by the cell types identified through ARCHS4 were used to build the ANKRD1 mesenchymal signature of 269 genes. e Uniform manifold approximation and projection (UMAP) of scRNA-seq in head and neck SCC (HNSCC). The clusters of the different cell types are as reported in ref. ⁴⁰. Red boxes indicate the fibroblasts population (left), overlapping with cells expressing ANKRD1 signature (right), 5902 cells from 18 patients were analyzed. f Pearson’s correlation analysis of ANKRD1 signature score and CAFs signature score (GSE122372) in the scRNA-seq of HNSCC shown in (e). g Spearman’s correlation between ANKRD1 expression and ANKRD1 signature in indicated patient’s cohorts derived from the TCGA database (LUSC = 485 patients, CESC = 297 patients, HNSC = 504 patients). h Kaplan-Meier (KM) survival analysis of indicated TCGA cohorts relative to ANKRD1 expression. Cox regression analysis. Results are adjusted for linear variables (age, sex, and stage). P values are indicated for each tumor cohort.