An FGFR-p53 developmental signaling axis drives salivary cancer progression

Abstract

Mucoepidermoid carcinoma (MEC) is the most frequently occurring salivary gland malignancy. Here, we investigated transcriptomic profiles of human fetal and adult salivary glands and MEC tumors to assess programs involved in MEC progression. Molecular and genetic analyses revealed that MEC tumors and fetal salivary glands share proliferative and developmental gene expression profiles that implicate an FGFR-p53 signaling axis in salivary MEC progression. Based on these findings, we developed a genetically engineered mouse model of advanced MEC via targeted expression of the CRTC1-MAML2 oncogene in salivary ductal cells. Specifically, CRTC1-MAML2 expression combined with p53 dysregulation in salivary ducts rewires FGF signaling to drive formation of tumors with histological and molecular features of high-grade MEC. The combined bioinformatics and mouse modeling of this study demonstrate that salivary MEC progression is underpinned by reactivation of developmental signaling programs and suggests a role for FGFR targeted therapies in the treatment of high-grade MEC.

Fig. 1. Fetal salivary glands and salivary MEC tumors share related transcriptional programs.

A DEG analysis of MEC tumor tissue vs normal adult parotid tissue. (Left) Volcano plot showing the relative expression of genes in each sample type. The dotted line represents P_adj = 0.010. Colored points above the line indicate DEGs with P_adj < 0.010. (Right) Quantification of the colored points from the volcano plot. The bar chart shows the number of upregulated (FC ≥ 2.00) and downregulated (FC ≤ −2.00) genes with P_adj < 0.010. B DEG analysis of fetal parotid tissue vs normal adult parotid tissue. (Left) Volcano plots showing the relative expression of genes in each sample type. The dotted line represents P_adj = 0.010. Colored points above the line indicate DEGs with P_adj < 0.010. (Right) Quantification of the colored points from the volcano plot. The bar chart shows the number of upregulated (FC ≥ 2.00) and downregulated (FC ≤ −2.00) genes with P_adj < 0.010. C Venn diagrams showing the number of DEGs identified in MEC tumors only (blue), fetal salivary glands only (red), and both (purple). Percentages reflect non-overlapping DEGs. D iRegulon-predicted transcription factor regulators of upregulated (left) and downregulated (right) DEG networks common to both MEC tumors and fetal salivary glands. E Hallmark GSEA pathway enrichment analysis showing upregulated and downregulated DEG pathways in MEC tumors vs adult parotid tissue. Only pathways with NES ≥ 1.50 or ≤−1.50 are displayed. Node size is proportional to the −log₁₀(padj) values. F Hallmark GSEA pathway enrichment analysis showing upregulated and downregulated DEG pathways in fetal vs adult parotid tissue. Only pathways with NES ≥ 1.50 or ≤−1.50 are displayed. Node size is proportional to the −log₁₀(padj) values. DEG differentially expressed gene, FC fold change, GSEA gene set enrichment analysis, MEC mucoepidermoid carcinoma, NES normalized enrichment score, NS not significant, SG salivary gland.

Fig. 2. High-grade salivary MEC tumors and developing fetal salivary glands are characterized by dysregulation of the p53 pathway.

A qPCR analysis of C1/M2 expression in low-grade and high-grade human MEC tumor samples. C1/M2 copy number per 10 ng input RNA was calculated based on a standard curve. Samples with ≥500 copies of C1/M2 per 10 ng input RNA were considered C1/M2-positive. Data is presented as the mean ± SD (n = 3 minimum technical replicates), excluding human MEC tumor samples where C1/M2 copy number was undetermined. B Left, the red and green fluorescent probes of the ZytoLight MAML2 Dual Color Break Apart Probe bind to the 3’ and 5’ ends of the MAML2 gene, respectively. Right, break apart MAML2 FISH showing a rearrangement involving MAML2 in a representative high grade MEC sample. Red and green arrows indicate location of single probe binding, with yellow arrow showing probe colocalization. Scale bar, 200 μm. C Principal component analysis of all parotid samples organized by collection site (UNC or UCSF), C1/M2 fusion status, and tissue origin and grade. D Venn diagrams showing the total number of overlapping DEGs in MEC tumors by grade number of upregulated (logFC ≥2.00) and downregulated (logFC ≤−2.00) genes. Genes altered in low-grade MEC, high-grade MEC, and fetal tissue are shown in the green, dark green, and yellow circles, respectively. The upper Venn diagram shows overlapping upregulated genes and the lower Venn diagram shows overlapping downregulated genes. E Log2 normalized counts of FOXM1 data generated via RNAseq. In the box and whisker plot, the horizontal line within the box represents the median, and the whiskers extend to ±1.5x the interquartile range (1.5*IQR). Wilcoxon-rank sum test of pairwise comparisons: ***P < 0.001; *P < 0.05; ns, not significant (P > 0.05). F Supervised cluster analysis of MEC tumor samples, adult salivary glands, and fetal salivary glands using the Troester et al. [98] 52-gene p53 signature. The fold change relative to the median expression value across all tumors is shown. The dendrogram branch is enriched for p53 mutant or p53 wild-type signatures (shown in magenta and gray, respectively). G Spearman correlation calculated for the Troester et al. [98] p53 signature (a composite of the wild-type and dysregulated signatures shown in panel F) in low-grade MEC (light green), high-grade MEC (dark green), fetal salivary tissue (yellow), and adult salivary tissue (purple). In the box and whisker plot, the horizontal line within the box represents the median, and the whiskers extend to ±1.5*IQR. Wilcoxon-rank sum test of pairwise comparisons: ***P < 0.001; *P < 0.05; ns, not significant (P > 0.05). DEG differentially expressed gene, FC fold change, C1/M2 CRTC1-MAML2, FOXM1 forkhead box protein M1, GSVA gene set variation analysis, HG high-grade, LG low-grade, MEC mucoepidermoid carcinoma, NES normalized enrichment score, ns not significant, PC1 principal component 1, PC2 principal component 2, qPCR quantitative polymerase chain reaction, RNAseq RNA sequencing, UCSF University of California at San Francisco, UNC University of North Carolina.

Fig. 3. Cell type-specific targeting of CRTC1-MAML2 to murine salivary glands identifies Krt14-positive basal epithelial progenitors that drive early ductal pathogenesis.

A Schematic representation of CreER/LoxP-mediated C1/M2 transgene expression in salivary glands. Left, Injection of transgenic animals with tamoxifen results in CreER activation, elimination of the STOP cassette, and subsequent expression of C1/M2 and the LumiFluor reporter in target cells. Right, The KRT14 promoter was used to express CreER in all salivary ductal cells, including the excretory, striated, and intercalated ductal cells, and myoepithelial cells. The Mist1 promoter was used to express CreER in all salivary acinar cells, including serous and mucous acinar cells. The Dcpp1 promoter was used to express CreER in the salivary intercalated ductal cells and the serous demilune cells. B Representative BLI images of control (LumiFluor allele with no CreER driver allele), Dcpp1-CreER;LumiFluor, Mist1-CreER;LumiFluor, and KRT14-CreER;LumiFluor animals 3 months and 6 to 12 months post-tamoxifen treatment. The scale bar indicates photons/sec. C qPCR analysis of C1/M2 expression in control (C1/M2-negative) (n = 8), Dcpp1-CreER;C1/M2 (n = 10), Mist1-CreER;C1/M2 (n = 4), and KRT14-CreER;C1/M2 (n = 4) animals up to 13 months post tamoxifen administration. C1/M2 transcript copy number per 10 ng input RNA was calculated based on a standard curve. Data is presented as the mean ± SD. D Representative H&E-stained submandibular glands from control (Mist1-CreER only), Dcpp1-CreER;C1/M2, Mist1-CreER;C1/M2, and KRT14-CreER;C1/M2 animals 6–9 months post tamoxifen administration. Scale bar indicates 200 μm at ×10 ×10 magnification. Inset images reflect ×60 ×10 magnification. BLI bioluminescent imaging, qPCR quantitative polymerase chain reaction, SLG sublingual gland, SMG submandibular gland.

Fig. 4. Dysregulation of p53 cooperates with C1/M2 to promote formation of tumors that share phenotypic hallmarks of human high-grade MEC.

A Schematic representation of KRT14-CreER/LoxP-mediated C1/M2 transgene expression coupled with Trp53 loss. B Left, representative KRT14-CreER;C1/M2;LumiFluor;Trp53^fl/fl mouse that developed a salivary gland tumor. Middle, the resected salivary gland tumor. Right, representative mucicarmine-stained tumor section. A defined border separates tumor tissue (left) from adjacent normal tissue (right). C qPCR analysis of C1/M2 expression in control (n = 8) and KRT14-CreER; LumiFluor;Trp53^fl/fl (KCT^fl/fl; n = 3) GEMM MEC tumor samples. C1/M2 copy number per 10 ng input RNA was calculated based on a standard curve. Data is presented as the mean ± SEM (n = 4 minimum technical replicates). D Overall survival of control (n = 5), KRT14-CreER;C1/M2 (KC; n = 4), KRT14-CreER;Trp53^fl/fl (KT^fl/fl; n = 3), KRT14-CreER;Trp53^fl/+ (KT^fl/+; n = 10), KRT14-CreER;C1/M2;Trp53^fl/+ (KCT^fl/+; n = 13), and KRT14-CreER;C1/M2;Trp53^fl/fl (KCT^fl/fl; n = 12) animals. E Quantification of total bioluminescent emission (photons/sec) from the salivary gland region in KRT14-CreER;LumiFluor;C1/M2;Trp53^fl/+ (KLCT^fl/+; n = 7), KRT14-CreER;LumiFluor;C1/M2;Trp53^fl/fl (KLCT^fl/fl; n = 10), and control (no KRT14-CreER allele; n = 3) animals, plotted as months post-tamoxifen induction. F Representative H&E-stained images of a control (LumiFluor;C1/M2) submandibular gland and a KRT14-CreER;C1/M2;Trp53^fl/fl MEC tumor. Scale bar: 200 μm. G Representative stained images from a control (LumiFluor;C1/M2) submandibular gland and a KRT14-CreER;C1/M2;Trp53^fl/fl MEC tumor. IHC staining was performed for p-AKT (S473), p-ERK1/2 (T202/Y204), Ki-67, and cleaved caspase-3. Scale bar: 200 μm. H Quantification of IHC H-scores in panel G. Data is presented as the mean ± SEM. I Venn diagram showing a comparison of DEGs captured by RNAseq of GEMM MEC tumors compared with human fetal salivary glands and low-grade and high-grade MEC tumors. BLI bioluminescent imaging, Cl Cas-3 cleaved caspase-3, CTRL control animals, DEG differentially expressed gene, GEMM genetically engineered mouse model, IHC immunohistochemistry, MEC mucoepidermoid carcinoma, PAS periodic acid-Schiff, qPCR quantitative polymerase chain reaction, RNAseq RNA sequencing.

Fig. 5. Alternative FGFR2 isoform usage establishes a pro-tumorigenic pathway in salivary MEC versus normal salivary glands.

A LogFC expression of FGF ligand and FGFR family members in human fetal salivary glands, low-grade MEC tumors, and high-grade MEC tumors compared to adult salivary gland tissue. B FGFR2b Reactome pathway analysis of high-grade MEC tumors. C Above, annotated illustration of the NCBI RefSeq gene reference for the two primary FGFR2 isoforms, curated subset (NM_* Annotation release 13 July 2013). Below, representative cigar plots of RNAseq data for FGFR2 visualized using the UCSC Genome Browser from adult salivary glands, fetal salivary glands, C1/M2-positive low-grade MEC tumors, and C1/M2 positive high-grade (HG) MEC tumors. D Quantification of FGFR2 exon 8 Percent Spliced In (PSI; Ψ) inclusion levels calculated by rMATS. Data represented as a violin plot with the mean as solid horizontal line and extending to the minimum and maximum values. E qPCR analysis of FGFR2 splice isoform expression in control and GEMM MEC tissues. ***P < 0.001; ns, not significant (P > 0.05). F Graphical schematic of proposed MEC progression cascade. The preneoplastic state is characterized by acinar disorganization, granular eosinophilia, and ductal disorganization upon acquisition of the t(11;19) translocation and C1/M2 expression. During the course of MEC oncogenesis, a shift in Fgfr2 isoform expression from the epithelial type, Fgfr2b, to a more mesenchymal form, Fgfrc marks the transition from a preneoplastic state to low-grade MEC. This receptor isoform switch is maintained in progression from low-grade to high-grade which is further characterized by p53 dysregulation, which we observed to be a feature shared with developing fetal salivary gland tissues. Generated using Biorender software. FC fold change, FGF fibroblast growth factor, FGFR fibroblast growth factor receptor, GEMM genetically engineered mouse model, HG high-grade, IgIII immune-globulin III domain, KD kinase domain, LG low-grade, MEC mucoepidermoid carcinoma, NES normalized enrichment score, Padj adjusted P value, qPCR quantitative polymerase chain reaction, RNAseq RNA sequencing, SG salivary gland, TM transmembrane domain.