Alternative lengthening of telomeres does exist in various canine sarcomas.

Alternative lengthening of telomeres (ALT) is a telomere maintenance mechanism (TMM) found in some human tumors such as sarcomas. Canine tumors are not characterized for ALT and the study aim was to identify if the ALT phenotype exists in canine sarcomas. Sixty-four canine sarcoma samples (20 snap-frozen, 44 FFPE) as well as six canine sarcoma cell lines were screened for ALT by C-circle assay. ALT was further evaluated by measuring telomere length via qPCR and telomere restriction-fragments including pulsed-field electrophoresis. ALT-associated proteins were validated by immunohistochemistry. Further, telomerase activity (TA) and gene expression were analyzed by TRAP and qPCR. DNA from 20 human neuroblastomas and 8 sarcoma cell lines served as comparative controls. ALT was detected in 9.4% (6/64) canine sarcomas including aggressive subtypes as hemangiosarcoma, osteosarcoma, and histiocytic sarcoma. C-circle levels were comparable with human ALT-positive controls. All ALT tumors demonstrated loss of ATRX expression and 5/6 showed strong p53 expression. TA was detected in 93% (14/15) snap-frozen samples including a sarcoma with ALT activity. This tumor showed long heterogeneous telomeres, and a high level of colocalization of DAXX with telomeres. One sarcoma was ALT and TA negative. All canine and human sarcoma cell lines were ALT negative. In this study, we demonstrated that canine sarcomas use ALT. As in humans, ALT was identified in aggressive sarcomas subtypes and coexisted with TA in one tumor. Overall, canine sarcomas seem to share many similarities with their human counterparts and appear an attractive model for comparative telomere research. © 2016 Wiley Periodicals, Inc.

Canine and human sarcomas exhibit predominant FGFR1 expression and impaired viability after inhibition of signaling.

Fibroblast growth factor receptors (FGFRs) are important in malignant progression of several human epithelial tumors. However, little is known about FGFRs in canine or human soft tissue sarcomas. Thus, our aim was to investigate expression of FGFRs and their involvement in cell survival in sarcomas of both species. FGFR1-4 and FGFRL1 transcripts as well as IIIb/IIIc splice variants of FGFR1-3 were evaluated in 3 canine- and 6 human sarcoma cell lines and 19 spontaneous canine sarcomas by SYBRqPCR. FGFR1 protein expression was assessed by immunohistochemistry. Growth inhibitory effects of FGFR1 inhibitor PD166866 and dominant negative recombinant FGFR adenoviral expression constructs (dnFGFR) on tumor cell lines were analyzed. Profiling of multiple FGFR transcripts detected comparable co-expression in most of human and canine sarcoma cell lines and canine tumor specimens. This indicates existence of closely related regulation mechanisms for FGFR expression in sarcomas of both species. FGFR1 with splice variant IIIc was consistently expressed with highest transcript levels. In 88% of the spontaneous tumor samples a heterogeneous FGFR1 protein expression was observed. Significant growth inhibition and cell death was seen after infection with dnFGFR1 in canine and human sarcoma cells, but not with dnFGFR3 and 4. PD166866 showed selective cytotoxicity with IC50 values between 12.1 and 26.4 µM. FGFR1 inhibition blocked ligand-induced tyrosine phosphorylation of ERK1/2 mitogen-activated protein kinase isoforms. This study emphasizes the important role FGFR1, especially splice variant IIIc, likely plays in sarcomas. Inhibitory small molecules could be of potential use for targeted therapy in aggressive sarcomas of both species.

Telomere Transcripts Target Telomerase in Human Cancer Cells.

Long non-coding transcripts from telomeres, called telomeric repeat-containing RNA (TERRA), were identified as blocking telomerase activity (TA), a telomere maintenance mechanism (TMM), in tumors. We expressed recombinant TERRA transcripts in tumor cell lines with TA and with alternative lengthening of telomeres (ALT) to study effects on TMM and cell growth. Adeno- and lentivirus constructs (AV and LV) were established for transient and stable expression of approximately 130 units of telomere hexanucleotide repeats under control of cytomegalovirus (CMV) and human RNase P RNA H1 (hH1) promoters with and without polyadenylation, respectively. Six human tumor cell lines either using telomerase or ALT were infected and analyzed for TA levels. Pre-infection cells using telomerase had 1%-3% of the TERRA expression levels of ALT cells. AV and LV expression of recombinant TERRA in telomerase positive cells showed a 1.3-2.6 fold increase in TERRA levels, and a decrease in TA of 25%-58%. Dominant-negative or small hairpin RNA (shRNA) viral expression against human telomerase reverse transcriptase (hTERT) results in senescence, not induced by TERRA expression. Population doubling time, cell viability and TL (telomere length) were not impacted by ectopic TERRA expression. Clonal growth was reduced by TERRA expression in TA but not ALT cell lines. ALT cells were not affected by treatments applied. Established cell models and tools may be used to better understand the role of TERRA in the cell, especially for targeting telomerase.