Alternative Telomere Lengthening and EMT in Cancer Stem Cell Depletion

Objectives: Telomeres are repetitive DNA-protein structures at chromosome ends maintained by telomeric repeat factor 2 (TRF2) and telomerase reverse transcriptase. We hypothesized that telomere dysfunction-induced stem cell depletion by deletion of TRF2 and telomerase would result in tumor resistance. The objective is to determine the effects of telomere dysfunction on stem cell phenotype in oral cancer in vivo.
Methods: Conditional double null K14Cre;TRF2f/f;Terc-/- and control mice were dosed twice weekly with 25 µg DMBA. Primary tumors were analyzed by histopathology. Epithelial mesenchymal transition (EMT) gene expression signatures were determined by microarray analysis. Telomeric DNA damage was determined by 53BP1 localization at the telomere. Downstream DNA damage signaling was determined by western blot. Telomere length was determined by quantitative metaphase fluorescence in situ hybridization (Q-FISH). Alternative lengthening of telomeres (ALT) was analyzed by expression of ALT associated PML bodies, telomeric circular DNA, and chromosome orientation-fluorescence in situ hybridization. K15+/CD34+ cancer stem cells were sorted by flow cytometry and localized by immunofluorescence microscopy.
Results: Surprisingly, 70% of oral cancers in K14Cre;TRF2f/f;Terc-/- mice exhibited histopathological features of EMT compared to 10% of control tumors. K14Cre;TRF2f/f;Terc-/- tumors expressed EMT genes Snail, Twist, and vimentin. Telomeres in K14Cre;TRF2f/f;Terc-/- tumors were characterized by increased DNA response, extreme shortening and high ATM/Chk2/p53 activation. Contrary to previous reports of stem cell expansion during EMT, the K15+/CD34+ cancer stem cell population was severely depleted due to DNA damage signaling. Cancer stem cell telomeres were maintained by the ALT pathway, a recombination based mechanism.
Conclusions: High levels of telomere dysfunction dramatically deplete K15+/CD34+ cancer stem cells. However, ALT induction maintains cancer stem cell telomeres and was associated with EMT. These results have implications for treatment outcomes in that telomerase inhibitor therapy may result in tumor progression due to ALT.