Telomerase Upregulation Induces Progression of Mouse BrafV600E-Driven Thyroid Cancers and Triggers Nontelomeric Effects

Abstract

Mutations in the promoter of the telomerase reverse transcriptase (TERT) gene are the paradigm of a cross-cancer alteration in a noncoding region. TERT promoter mutations (TPM) are biomarkers of poor prognosis in cancer, including thyroid tumors. TPMs enhance TERT transcription, which is otherwise silenced in adult tissues, thus reactivating a bona fide oncoprotein. To study TERT deregulation and its downstream consequences, we generated a Tert mutant promoter mouse model via CRISPR/Cas9 engineering of the murine equivalent locus (Tert-123C>T) and crossed it with thyroid-specific BrafV600E-mutant mice. We also employed an alternative model of Tert overexpression (K5-Tert). Whereas all BrafV600E animals developed well-differentiated papillary thyroid tumors, 29% and 36% of BrafV600E+Tert-123C>T and BrafV600E+K5-Tert mice progressed to poorly differentiated cancers at week 20, respectively. Tert-upregulated tumors showed increased mitosis and necrosis in areas of solid growth, and older animals displayed anaplastic-like features, that is, spindle cells and macrophage infiltration. Murine TPM increased Tert transcription in vitro and in vivo, but temporal and intratumoral heterogeneity was observed. RNA-sequencing of thyroid tumor cells showed that processes other than the canonical Tert-mediated telomere maintenance role operate in these specimens. Pathway analysis showed that MAPK and PI3K/AKT signaling, as well as processes not previously associated with this tumor etiology, involving cytokine, and chemokine signaling, were overactivated. These models constitute useful preclinical tools to understand the cell-autonomous and microenvironment-related consequences of Tert-mediated progression in advanced thyroid cancers and other aggressive tumors carrying TPMs.

Implications: Telomerase-driven cancer progression activates pathways that can be dissected and perhaps therapeutically exploited.

Figure 1.

Modeling of Tert upregulation in genetically engineered mouse models with endogenous Braf^V600E expression. A, Schematic representation of the sequence alignment of human TERT (top) and mouse Tert promoter (bottom) sequences. A single C>T transition at the conserved human c.−124C and mouse c.−123C loci generates a consensus binding motif for Ets transcription factors (“Ets cons”). B, Tert promoter-driven luciferase expression, normalized by renilla (“Luc/Ren”), in mouse cell lines using Tert wild-type (mTert −123C, green) and mutant (mTert −123T, red) constructs. C, Genetic schema of the mouse models: Top, Thyroid-specific expression of Cre recombinase [driven by thyroid peroxidase (TPO) in TPO-Cre constructs] substitutes exon 15 of WT Braf by V600E mutant allele, resulting in endogenous expression of Braf oncoprotein (66). Cre-mediated excision of stop cassette also enables YFP expression in thyroid cells; Bottom: Tert^−123C>T is knocked-in in the germline via CRISPR/Cas9 editing of mouse zygotes, as described (left); mouse Tert is driven by the keratin 5 (K5) promoter (middle; ref. 17); breeding of LsL-Braf, TPO-Cre/eYFP, and Tert alleles generates the three genotype combinations used in this study (right).

Figure 2.

Histologic analysis of the Braf+Tert mouse models. Representative H&E-stained thyroid sections of Braf^V600E, Braf^V600E+Tert^−123C>T, and Braf^V600E+K5-Tert mice at (A) 6 to 12 weeks (all displaying PTC phenotypes); and (B) 20–30 weeks. In B, the second panel represents a PDTC fulfilling the Turin criteria (solid growth and high mitotic index), whereas the third and fourth panels show tumors within the newly defined DHGTC category. C, Percentage of animals with the indicated genotypes showing PTC or PDTC/DHGTC at 6 to 12 weeks (left) and 20 to 30 weeks (right). D, Examples of 40-week Braf^V600E (top) and Braf^V600E+Tert^−123C>T tumors (middle and bottom). Some Braf^V600E animals at this age develop signs of PDTC transformation (top) and typically retain Pax8 expression (top, right). Only Braf^V600E+Tert^−123C>T animals of this age developed tumors with ATC-like features within a PTC component (middle and bottom), overall loss of Pax8 with spindle cells retaining Pax8 positivity (middle, right), spindle cells (bottom, center, yellow arrows), and positivity for F4/80 stain, denoting macrophage infiltration (bottom, right). E, Example of a 20-week Braf^V600E+K5-Tert tumor with similar features than tumors from E, that is, ATC-like areas, spindle cells (yellow arrowhead) with focal positivity for Pax8 (spindle cells with red arrowheads). “Braf+Tert” and “Braf+K5-Tert” labels denote “Braf^V600E+Tert^−123C>T” and “Braf^V600E+keratin 5-driven Tert” genotypes, respectively.

Figure 3.

Characteristics of Tert re-expression in telomerase-upregulated thyroid cancers. A, Relative levels of Tert transcription in YFP-sorted cells isolated from Braf^V600E+Tert^−123C>T mouse tumors, showing an increase in Tert mRNA levels for specimens collected at 20 weeks versus 10 weeks. Each red point represents a tumor collected from a different animal at the indicated age. Results are expressed as fold change compared with the Tert baseline expression of Braf^V600E tumors from the same ages (dotted blue line). B, Representative examples of thyroid specimens from 20-week animals with the indicated genotypes subjected to RNAscope to detect Tert single mRNA molecules (green dots). DAPI (blue) is used for contrast. Red squares denote areas for which higher magnifications are provided. C, Quantification of number of Tert transcripts at single-cell resolution from RNAscope data on mouse tumors with the indicated genotypes. Data are represented as percentage of cells within each tumor expressing 1, 2, 3, 4, or 5+ transcripts. Tumor boundaries were defined manually and cell detection and quantification of green fluorescent dots was performed employing the built-in automated analysis tools on QuPath, using the same detection parameters across specimens. D, Relative mRNA levels in cell lines derived from Braf^V600E (n = 4), Braf^V600E+Tert^−123C>T (n = 5), and from Braf^V600E+K5-Tert (n = 3) mouse tumors, showing that genotype-dependent increases in Tert transcription are maintained in vitro. E, Mean nuclear telomeric intensity, measured in arbitrary fluorescence units (auf) via Q-FISH (quantitative FISH) in Braf^V600E (n = 6), Braf^V600E+Tert^−123C>T (n = 10) and Braf^V600E+K5-Tert (n = 7) mouse primary tumors derived from 20-week animals. “Braf+Tert” and “Braf+K5-Tert” labels denote “Braf^V600E+Tert^−123C>T” and “Braf^V600E+keratin 5-driven Tert^” genotypes, respectively.

Figure 4.

Transcriptomic characterization of telomerase-upregulated mouse thyroid tumors. A, Schematic representation of the isolation of thyroid tumor cells for RNA-seq. B, Comparison of RNA-seq normalized counts for Tert in pooled thyroid glands with the indicated genotypes. Values represent average expression levels for pooled WT (n = 25), Tert^−123C>T (n = 17), and K5-Tert (n = 14) animals. C, Comparison of RNA-seq normalized counts for Tert in thyroid tumors from Braf^V600E (n = 4), Braf^V600E+Tert^−123C>T (n = 7), and Braf^V600E+K5-Tert (n = 4) animals. Each dot represents an individual mouse. D, Volcano plot showing significantly under- and overexpressed genes in Braf^V600E+K5-Tert tumors compared with Braf^V600E. Specific genes are indicated in red. E, Top 10 upregulated terms from the KEGG pathway database analysis for the Braf^V600E versus Braf^V600E+K5-Tert comparison. F, Top 10 upregulated terms from the GSEA hallmarks of cancer analysis for the Braf^V600E versus Braf^V600E+K5-Tert comparison.

Figure 5.

Validation of telomerase-upregulated pathways. A, Western blot analysis using protein extracts from thyroid tumors from 20-week-old animals with the indicated genotypes for phospho-NF-κB p65 and vinculin (VCL, loading control). B, Representative images from phospho-Erk (pErk) IHC performed in mouse tumors from 20-week-old animals with the indicated genotypes. C, Quantification of the percentage of tumor cells staining positive for pErk in mice with the indicated genotypes. D, Western blot analysis using protein extracts from thyroid tumors from 20-week-old animals with the indicated genotypes for phospho- and total Erk (MAPK pathway) and Akt (PI3K effector), as well as p85 (loading control; only protein that was reblotted on stripped membranes). “Braf+Tert” and “Braf+K5-Tert” labels denote “Braf^V600E+Tert^−123C>T” and “Braf^V600E+keratin 5-driven Tert” genotypes, respectively.